Chronic liver disease (CLD) in children constitutes a major health burden on both parents and the child with the disease. They may be caused by infectious, autoimmune, metabolic, vascular, drugs, and toxins or unidentified etiologies. Many of these CLDs progress toward cirrhosis and eventually liver failure. Although some of these disease categories are subjected to specific treatment with a good prognosis, several are not, especially when there is no identifiable etiology 1.
The liver regulates the metabolic pathways and transport of trace elements, and consequently their bioavailability, tissue distribution, and eventual toxicity. The liver also plays a role in the excretion of trace elements through bile formation 2. Growing evidence indicates that many trace elements play important roles in a number of biological processes by activating or inhibiting of enzymatic reactions, competing with other elements and metalloproteins for binding sites, and affecting the permeability of cell membranes 3,4. Moreover, some trace elements such as zinc (Zn), iron (Fe), and copper (Cu) exert important protective or enhancing effects on the progression of some diseases 5.
Zn is an essential and the most abundant intracellular trace element that plays a central role in cellular growth and differentiation. After ingestion, Zn is initially transported to the liver and then distributed throughout the body. The organ with the highest initial Zn uptake is the liver. Thus, in patients with liver disease, especially in those with cirrhosis, Zn deficiency can occur. It is a common cofactor of various enzymatic systems, including the ammonium metabolism and urea cycle, which occurs in the liver 6.
Zn is involved in stabilizing the cell membrane and prevents oxidative destruction caused by free radicals. The antioxidant actions of Zn include the induction of metallothionein (Zn-binding protein, formed by the liver), which is a potent scavenger of toxic metals and hydroxyl radical 7. Fe and Cu ions catalyze the production of hydroxyl radical from hydrogen peroxide (H2O2). Zn is known to compete with both Fe and Cu for binding to the cell membrane, thus decreasing the production of hydroxyl radicals 8. Thus, it is clear that Zn plays multiple roles as an antioxidant and is therefore an excellent candidate for clinical chemoprevention trials in humans 2. Serum Zn levels among children with CLD have not been fully investigated in a large number of children, and thus, Zn is not a part of the recommended micronutrient intake for these patients. It is, similarly, unknown whether there is an association between the Zn status and the severity of liver disease 9.
Cu is a trace element that is essential for the growth and differentiation of cells. However, it is highly toxic in excess and results in cellular damage 4,10. It functions as a cofactor in various redox reactions and the formation of deleterious free radicals is enhanced by the presence of Cu ions 11.
Fe is required for many enzymes that are critical for cellular function to the extent that there is doubt that any form of life exists in the absence of Fe. It also plays a fundamental role in oxygen-carrying proteins such as hemoglobin and myoglobin. However, Fe can be toxic when present in excess as it is able to catalyze the formation of reactive oxygen species. Highly specialized proteins have been developed for efficient extracellular transport (transferrin) and intracellular storage (ferritin) of Fe 12.
Although many studies have been carried out for the assessment of trace elements in adults with CLD 2,4,13–17, very few studies have focused on children 9,10. Therefore, we aimed to measure serum levels of essential trace elements in children with CLD irrespective of the etiology and correlate these serum levels with biochemical measures of liver damage, transaminases, and other liver function tests.
Materials and methods
This study included 50 children with CLD. They were recruited from among the attendants of the outpatient and inpatient clinic of the Department of Pediatric Hepatology, National Liver Institute, Menoufiya University, from October 2010 to February 2012 on a consecutive basis. Another group of 50 age-matched and sex-matched healthy children were enrolled as a control group. None of the participants had received mineral supplements before blood sampling. Also, those with recent blood loss or transfusions were excluded. A written informed consent was signed by the parents of each child. The study was approved by the Research Ethics Committee of the National Liver Institute, Menoufiya University.
After full assessment of history, thorough clinical examination, and routine and specific investigations according to the expected etiology, the etiological diagnoses of the CLD group were autoimmune hepatitis (n=7), chronic hepatitis C (n=5), Alagille syndrome (n=5), cytomegalovirus hepatitis (n=4), progressive familial intrahepatic cholestasis (n=3), neglected biliary atresia (n=3), chronic hepatitis B (n=2), glycogen storage disease (n=2), Crigler–Najjar syndrome (n=2), congenital hepatic fibrosis (n=2), Niemann–Pick disease (n=2), inspissated bile syndrome (n=2), toxoplasma hepatitis (n=2), Caroli syndrome (n=2), portal vein thrombosis (n=2), Wilson’s disease (n=1), Dubin–Johnson syndrome (n=1), Budd–Chiari syndrome (n=1), α1-antitrypsin deficiency (n=1), and cystic fibrosis (n=1).
Under complete aseptic precautions, 10 ml venous blood was withdrawn into a vacutainer tube and allowed to clot naturally, then centrifuged and the clear supernatant serum was separated and divided into two aliquots. One aliquot was assayed for liver function tests and the other was stored at −20°C until the assay of trace elements. Liver function tests (alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), γ-glutamyl transpeptidase (GGT), total bilirubin, direct bilirubin, total proteins, and serum albumin) were performed using Automated Beckman Coulter (Beckman Coulter Inc., Brea, California, USA). Serum Zn, Cu, and Fe were measured using a colorimetric assay at a wave length of 560 nm (for Zn), 580 nm (for Cu), and 546 nm (for Fe) using the Biosystems BTS-310 photometer chemistry analyzer (Biosystem S.A., Barcelona, Spain). TIBC was measured using the ferrozine method by Spectrum Diagnostic for the in-vitro quantitative, diagnostic determination of TIBC in human serum. Serum ferritin was measured by enzyme-linked immunosorbent assay using the UBI Magiwel Ferritin Quantitative device (United Biotech, Inc., Mountain View, California, USA) and serum transferrin saturation (TS) was calculated by dividing serum Fe by TIBC 13.
Descriptive results were expressed as mean±SD or number and percentage. For qualitative data, significance was tested using the χ2-test. For quantitative data, the significance between two groups was tested using Student’s t-test for parametric data and using the Mann–Whitney U-test for nonparametric data. Correlation was tested using Pearson’s correlation. Results were considered significant at P-values less than 0.05. Statistical analysis was carried out using the SPSS (statistical package for social science) program, version 13 (SPSS Inc., Chicago, Illinois, USA) on an IBM-compatible computer.
Characteristics of the study population
The current study included a group of 50 children with CLD (27 males and 23 females, mean age 5.86±4.76 years) and another group of 50 age-matched and sex-matched (P>0.05 for both) healthy children as controls (25 males and 25 females, mean age 6.62±3.54 years). We found that liver enzymes (AST, ALT, ALP, GGT) and total and direct bilirubin were significantly higher in the CLD group than that in the control group, whereas albumin and total protein were significantly lower in the CLD group than that in the control group (P<0.01) (Table 1).
Trace elements’ serum levels in the studied groups
Serum Zn and TIBC were significantly lower in the CLD group than that in the control group, whereas serum Cu, Fe, ferritin, and TS were significantly higher in the CLD group than that in the control group (P<0.01) (Fig. 1).
Correlation of trace elements with disease severity
Serum Zn had a significant negative correlation with serum Cu, Fe, AST, and ALT (P<0.01 for all) (Fig. 2). However, serum Cu was significantly positively correlated with each of Fe, AST, ALT, GGT, and total and direct bilirubin (P<0.01 for all) (Fig. 3). In addition, there was a significant positive correlation between Fe and each of AST and ALT (P<0.01 for both) (Fig. 4). Moreover, there was a significant positive correlation between ferritin and AST (P<0.05) (Fig. 5), whereas there were no significant correlations between trace elements and other liver function tests (Figs 2–5).
CLDs in children are associated with significant morbidity and mortality in the absence of successful treatment. One of the obstacles against their successful management is insufficient knowledge of the factors promoting disease progression 1. Therefore, considerable efforts have been directed toward identification of factors aggravating or improving liver damage in those with CLD.
Trace elements especially those involved in the antioxidant system such as Zn or those with oxidative properties, such as Cu and Fe, may play a role in pathological progression of CLD as they may have a direct hepatic toxicity (Cu and Fe) or may be decreased as a consequence of the impaired liver function (Zn) 18,19.
In this study, serum Zn level was significantly lower in children in the CLD group than the control group (68.64±19.47 vs. 95.92±8.77 μg/dl, P<0.01). This result is in agreement with that of Umusig-Quitain and Gregorio 9, who found that children with CLD, whether in a compensated or a decompensated state, had lower serum Zn levels compared with healthy controls (68.07±31.55 vs. 89.9±25.9 μg/dl, P<0.0001), and as the severity of liver disease worsened, Zn levels decreased. Furthermore, in studies carried out in an adult population, it was found that cirrhotic patients showed a significant decrease in serum Zn compared with the controls 4,14,17. In addition, Gur et al. 16 reported that hepatic Zn concentrations were below normal in patients with liver cirrhosis because of hepatitis B virus infection. Moreover, Kolachi et al.2 found that the Zn level was lower in patients with liver cirrhosis/cancer compared with healthy individuals (P<0.001).
Zn is transported in blood plasma mostly by albumin (60–70%) and by α2-macroglobulin (30–40%) 20. In our study, the significantly lower serum Zn level we observed in children with CLD compared with controls might be the result of the decreased serum albumin level, decreased α2-macroglobulin synthesis, poor dietary intake, or protein restriction. Because of this finding, the intake of Zn supplementation should be encouraged for children with CLD. However, other trace elements that can interfere with Zn absorption, such as Cu and Fe, should also be investigated among these children.
In the present study, serum Zn showed a significant negative correlation with serum Cu, Fe, AST, and ALT (P<0.01). The negative correlation between Zn and each of Cu and Fe can be attributed to the rationale that elements with similar physical or chemical properties will act in antagonism to each other biologically. Such metals could compete for binding sites on transporter proteins or on enzymes requiring metals as cofactors. The intestinal competition of Zn with Cu and Fe has been considered as a prime example of competitive biological interactions between metals with similar chemical and physical properties 21,22. The significant negative correlation between serum Zn and the biochemical parameters of hepatocyte necrosis and liver damage in our study (ALT and AST) may reflect the presumed protective role of Zn, being an antioxidant, against progression of liver disease irrespective of the primary etiology.
It has long been speculated that Zn exerts a protective effect against liver fibrosis. Zn supplementation in cirrhotic patients has beneficial effects on liver metabolism 23 as it is associated with decreased oxidative stress and improved immune function, and might aid in the prevention and treatment of cancer. After Zn supplementation for 60 days in cirrhotic adults, there was an improvement in liver function in terms of increased albumin and decreased bilirubin, ALP, GGT, and ferritin 2. In rats, Zn supplementation inhibited hepatic cellular damage induced by exposure to an electromagnetic field 24. Accordingly, Zn supplementation should be encouraged for all Zn-deficient CLD children and more studies on the effects of this supplementation on liver function and liver disease progression are recommended.
We found that serum Cu level was significantly higher in children in the CLD group than that in the control group (149.0±17.85 vs. 94.84±10.97 µg/dl, P<0.01). This finding is in agreement with that of Rahelic et al. 14, who found that serum concentration of Cu was significantly higher in patients with liver cirrhosis (P<0.001) than healthy controls. Also, Sayed et al.19 found similar results in a study on patients with CLD in the population of a rural area in Egypt.
In the present study, serum Cu was positively correlated with Fe, AST, ALT, GGT, and total and direct bilirubin (P<0.01 for all). The fact that the metabolism of Cu and Fe is interlinked has been known long time ago. Divalent metal transporter-1 (DMT1) is a transporter responsible for intestinal Fe uptake. Electrophysiological evidence suggests that DMT1 can also be a Cu transporter 22. Cu is highly toxic in excess and results in cellular damage 21. It can bind to proteins and nucleic acids and cause the oxidation of lipids and proteins by enhancing the formation of deleterious free radicals 11.
Our present findings of significantly higher serum Cu in children with CLD than that in the controls (P<0.01) and its positive correlation with each of Fe, AST, ALT, GGT, and total and direct bilirubin (P<0.01 for all), in conjunction with the oxidative and malignant potentials initiated by Cu 25, suggest that high Cu levels in children with CLD might lead to a more severe outcome and that a low-Cu diet might be necessary for CLD children irrespective of the etiology. This rationale is supported by the study of Goodman et al.25, who reported an anticancer strategy through Cu deficiency because of its role in promoting physiological and malignant angiogenesis.
The liver is an important organ in Fe homeostasis. Besides its involvement in Fe storage, the liver also produces transferrin and hepcidin, a Fe carrier protein in plasma and a hormone regulating Fe metabolism, respectively 26. Another aspect of the relationship between Fe and the liver is that this organ is one of the main targets in hemochromatosis. Following the model of hereditary hemochromatosis, the possible role of Fe overload as a cofactor for disease progression in acquired liver diseases has been investigated, with controversial results 27. Serum Fe, TIBC (i.e. transferrin activity), and ferritin levels are the principal tests used in the evaluation of Fe burden. Another frequently used parameter, TS, shows the percent saturation of transferrin. Serum Fe parameters may be affected by liver disorders, and it is believed that these tests are unreliable in CLD 13.
Our results showed that serum Fe, ferritin, and TS were significantly higher in children with CLD than that in healthy controls (P<0.01). However, serum TIBC was significantly lower in the CLD group than that in healthy controls (P<0.01). Moreover, there was a positive correlation between Fe and AST and ALT (P<0.01 for both). Finally, there was a positive correlation between ferritin and AST (P<0.05).
In a similar study carried out in adults with CLD by Jurczyk et al. 28 it was found that the mean value of serum Fe concentration ranged from 144 to 160.9 µg/dl, with no significant difference among the disease groups studied (P>0.05). The TIBC was significantly lower in alcoholic cirrhosis of the liver (268.3 µg/dl) in comparison with chronic hepatitis C (P<0.004) and chronic hepatitis B (P<0.04). TS was significantly higher in alcoholic cirrhosis compared with both chronic hepatitis C (P<0.0031) and chronic hepatitis B (P<0.024). Moreover, serum ferritin was significantly higher in cirrhotic patients, irrespective of the etiology, compared with patients with chronic viral hepatitis (P<0.045). Another study reported that serum ferritin and TS were significantly higher in patients with cirrhosis and cancer (P<0.001) 2. However, Lin et al.4 found that there was no significant difference in serum Fe between chronic hepatitis B adult patients and controls, whereas there was a significant decrease in those with hepatocellular carcinoma than that in healthy controls.
Our correlation studies for serum Fe and ferritin with biomarkers of liver damage, liver enzymes, were in agreement with those of Jurczyk et al.28. They found a correlation between an increase in both AST and ALT and a higher level of ferritin in patients with chronic hepatitis C (P<0.005 and P<0.02, respectively), chronic hepatitis B (P<0.05 and P<0.03, respectively), and alcoholic hepatitis (P<0.05 for both). It has been proposed that Fe plays a significant role in progression of liver fibrosis in viral hepatitis, and serum Fe parameters, especially ferritin level, might reflect hepatic Fe accumulation 29.
There is growing evidence that mildly increased amounts of liver Fe can increase hepatic injury. The rationale that makes Fe a potential hepatotoxic factor is based on the capability of this metal to induce oxidative stress by catalyzing the formation of hydroxyl radicals through the Fenton reaction. Fe-catalyzed oxidative stress causes lipid peroxidation and protein modification, DNA damage, with consequent promotion of mutagenesis, and leads to the exhaustion of antioxidant defenses, so that the term ‘ferrotoxic diseases’ has been coined 30.
This concept could be supported by our results of a positive correlation of the oxidant trace elements Cu, Fe, and ferritin with biomarkers of liver damage and, in contrast, the negative correlation of the antioxidant Zn with biomarkers of liver damage. In agreement with our results, and supporting this concept, is the study of Kolachi et al. 2. They found that after Zn and selenium supplementation for adults with CLD, the ferritin levels were decreased from 240±28.8 to 158±12.6 ng/ml and TS decreased from 52.5±2.9 to 45.9±3.3%. Moreover, this was associated with an improvement in liver functions in terms of increased serum albumin and decreased serum bilirubin, ALT, AST, ALP, and GGT.
Prospective, randomized studies are required to evaluate whether Fe and Cu depletion and Zn supplementation may reduce liver damage, fibrosis progression, and eventually morbidity and mortality in children with CLD.
Our finding of significantly higher serum Fe, Cu, and ferritin in children with CLD than that in healthy controls and their positive correlation with biochemical parameters of liver damage together with the significantly lower serum Zn in CLD than that in healthy controls and its negative correlation with biochemical parameters of liver damage encourage the inclusion of these trace elements as biomarkers for monitoring the severity of liver damage during routine assessment of children with CLD. Finally, we recommend caution to avoid excess Fe and Cu intake in such patients, whereas Zn supplementation should be encouraged irrespective of the etiology of CLD.
This study was funded by the National Liver Institute, Menoufiya University, Egypt, without any particular role in the study design, recruitment of individuals, data analysis, or the writing of the manuscript.
Conflicts of interest
There are no conflicts of interest.
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