Hippokratia 2014, 18(2):144-149
Papanikolopoulos K1, Alexopoulou A1, Dona A2, Hadziyanni E1, Vasilieva L1, Dourakis S1
12nd Department of Internal Medicine, Hippokration General Hospital, 2Department of Forensic Medicine and Toxicology, Medical School, University of Athens, Athens, Greece
Background: Copper (Cu) and Zinc (Zn) are essential trace elements which play an important role in various biological processes. Zn deficiency is common in liver diseases while Cu deficiency is rarely reported. To determine whether serum Cu and Zn concentrations differed in acute hepatitis, compared to controls and investigate possible correlations of Cu and Zn values with etiology and severity of liver diseases.
Methods: Serum Cu and Zn concentrations were determined by air acetylene flame atomic absorption spectrometer in 40 patients (acute hepatitis A, B, C, autoimmune and drug induced hepatitis) and 150 healthy controls.
Results: Compared to healthy controls, significantly higher Zn levels were found in patients (106.5 µg/dl, P <0.01). Abnormal levels of either Cu and/or Zn were found in 48% of patients vs 23.3% of the controls (P =0.01). Ten patients had abnormal Zn and fourteen had abnormal Cu levels. There was a trend for the severe hepatitis cases to have abnormal Cu values and in this subgroup Cu and Zn were positively correlated with prothrombin time and alanine aminotransferase (ALT) levels, respectively. Cu and Zn levels did not differ statistically across groups of different etiologies.
Conclusions: Abnormalities in Cu and Zn concentrations are common in acute hepatitis. Cu and Zn exhibited positive correlations with prothrombin time and ALT respectively, in severe cases.
Key words: Trace elements, viral hepatitis, autoimmune hepatitis, drug-induced hepatitis, cryptogenic hepatitis
Corresponding author: Konstantinos P. Papanikolopoulos, M.D., 30 Aeroporou Papanastasiou, 11527 Athens, Greece, tel: +302107700202, e-mail: firstname.lastname@example.org
Copper (Cu) and Zinc (Zn) are essential trace elements as they act as cofactors of antioxidant enzymes to protect the body from oxidative stress1,2. In particular, Cu is a component of many important enzymes, including amine oxidases, ferroxidases, cytochrome c-oxidase, dopamine b- hydroxylase, superoxide dismutase and tyrosinase1. Zn contributes to zinc finger proteins that interact with DNA and approximately 250 proteins contain Zn including DNA polymerase, RNA polymerase and transfer RNA synthetase3,4. Intestinal absorption of Zn occurs by a specific process that is enhanced in pregnancy and by corticosteroids while diminished by co– ingestion of phytates, phosphates, iron, Cu, lead and calcium5,6.
A large number of studies have shown that Cu and Zn are implicated in cardiovascular, autoimmune, cancer and degenerative diseases7-9. During most viral infections, the plasma levels of trace elements change10 and their effect has been studied on a variety of infectious diseases11. The changes in trace element levels are induced by cytokines in response to several stimuli, including stress and infection12.
Although there are studies about the association of trace elements with acute13,14 or chronic viral hepatitis15,16, there is no data about the above trace elements concerning other types of acute liver diseases (autoimmune, drug- induced, acute hepatitis B, cryptogenic) and in particular, the association of their values with the severity of the disease.
The aim of this study is to determine possible disturbances of Cu and Zn levels in patients with acute liver diseases and investigate correlations with etiology and severity of liver disease.
Forty patients (21 men and 19 women) with acute hepatitis (ALT > 400 U/L) aged between 18 and 80 years, were evaluated in the outpatient Clinics or hospitalized in the 2nd Department of Medicine, in the time interval 2009 and 2011. One hundred fifty healthy blood donors (75 men and 75 women, in the age range 18-70 years) were included in the study with an approximate matching by age group and men/women ratio (Table 1). None of the controls received any medication. The study was approved by the Hospital Ethics Committee. Half of the patients had acute viral hepatitis (fourteen acute hepatitis B, three acute hepatitis Α and three acute hepatitis C), five patients drug-induced, eight autoimmune and seven cryptogenic. Severe cases were defined as those with prothrombin time > 15 sec and/or albumin < 3.5 g/dl. All severe hepatitis cases were icteric and had a total bilirubin level more than 10 mg/dl.
Based on the available information, that is a ratio of approximately ¼ for cases and controls and assuming a proportion of 15% with abnormal values for Cu or Zn in the control group (Kouremenou-Dona et al, 2006)17, we calculated a power of nearly 0.60, 0.80 and 0.90 to detect a 15, 20 and 25% higher proportion of abnormal values in the patients group considering a type I error of 0.005.
Serum samples and analytical methods
Demographics and liver biochemistry [aspartate aminotransferase (AST), ALT, alkaline phosphatase (ALP) and bilirubin], prothrombin time and albumin values were recorded at the first visit of the patient group. The etiology of acute hepatitis of viral etiology was identified as follows: Cases of acute hepatitis B were positive for IgM anti-HBc and in the vast majority the source of infection was identified; cases of acute hepatitis A were positive for IgM anti-hepatitis A virus (HAV) antibody; cases of acute hepatitis C had a recent seroconversion from anti-Hepatitis C virus (HCV) negative to anti-HCV positive status and detectable HCV RNA. A score ≥ 7 (definite) was used for the diagnosis of autoimmune hepatitis according to Simplified criteria for the diagnosis of autoimmune hepatitis18. In cryptogenic hepatitis cases, viral markers, autoantibodies and drug history were negative, IgG levels were normal and metabolic liver diseases as Wilson disease or hemochromatosis were excluded. All cryptogenic cases had a liver biopsy without any specific characteristics. Drug-induced acute hepatitis cases reported an administration of a suspect drug with temporal association to the illness.
Serum samples at the first visit were centrifuged and stored at 18°C. All glassware and bottles used for the isolation and analysis of serum were previously soaked in diluted nitric acid (10%) for 3 hours and rinsed thoroughly with de-ionized water. This procedure was followed in order to exclude the possibility of contamination with Zn or Cu. All samples were diluted (1:4) using water. The determination of Cu and Zn was carried out in serum samples by air acetylene flame atomic absorption spectrometer (model Spectra AA, Varian, Australia) equipped with D2 lamp background correction system. A Cu and Zn hollow cathode lamp (Varian) operating at 10 mA intensity and a spectral width of 0.7 nm was selected to isolate the 324.7 nm and 213.9 nm lines for Cu and Zn respectively. All analyses were performed in peak height mode to calculate absorbance rates. All samples were analyzed in triplicate.
Serum Cu and Zn values were expressed in μg/dl. Based on the Kouremenou-Dona’s et al (2006)17 work, we considered as reference values those between 70 and 155μg/dl for Cu and between 60 and 150μg/dl for Zn. The trace elements were measured in the Department of Forensic Medicine and Toxicology, Medical school, University of Athens.
All analyses were performed using the Stata statistical package (Stata Corporation: Stata/SE 11.0 for Windows, Lakeway Drive, College Station, TX, USA, 2009). For descriptive statistics, results are presented as number and percentage for categorical variables and mean and standard deviation for continuous variables. The median and inter-quartile range was estimated for Cu and Zn and the non-parametric test of equality of medians was applied to test differences in serum measurements between cases and controls or between men and women. The Chi square and Fisher’s exact tests were used to compare the differences in the prevalence of abnormal values for Cu and zinc among cases and controls. The Spearman’s rank correlation coefficients between Cu and zinc with several clinical measurements were subsequently estimated for the patients group only. Significance was generally tested at the 5% level of statistical significance (p < 0.05).
Characteristics of patients and controls are presented in Table 1. The distribution of cases and controls across the age groups was generally equal. Gender distribution was also equal between patients and controls.
The median values and first and third quartiles for Cu and Zn are presented in Table 2 for both patients and healthy individuals, overall, by sex and age group. Levels of Cu in patients were similar to controls. For zinc, higher values were found among patients overall (p<0.01) and among men (p =0.01) and women (p =0.03) separately, when compared to controls (p1). Cu and Zn values were similar between men and women in acute hepatitis group (p2).
The distribution of patients and healthy individuals with normal and abnormal values for both trace elements are depicted in Table 3. The normal values were based on data of previous investigators from the same area17. Nearly half of the cases had abnormal values either for Cu and/or for Zn (47.5%). The corresponding percentage was much lower in controls (23.3%) (p=0.01). Most of the analysed sample for healthy individuals lied within the reference intervals (approximately 90% for Cu and 85% for Zn), while the proportions of abnormal values in both trace elements were significantly higher among cases (35% for Cu and 25% for Zn, p <0.01 and p =0.15 respectively) than among controls. Eight patients had Zn levels above normal and two below normal. Nine patients had Cu levels above normal and five below normal.
The results for Cu were more striking among men and that for zinc among women.
The analysis of the proportions of patients who had abnormal values of trace elements according to the cause of acute hepatitis showed that similar frequencies of patients with abnormal values of Cu or Zn were found across different etiologies of acute hepatitis (Table 4).
The values of both trace elements according to the severity status showed that both Cu and Zn were higher in severe than in non-severe cases and the proportions of patients with abnormal values showed that there was a trend for the severe cases to have abnormal Cu values (47.6% vs 21%) without any of the above results gaining a statistical significance (Table 5).
Table 6 presents the spearman’s rank correlation coefficients of Cu and Zn with several laboratory measurements across patients. In the total of patients no significant correlations were found. However, Cu values were positively correlated with prothrombin time (r=0.46) and Zn were positively correlated with ALT (r=0.45) in the subgroup of severe acute hepatitis cases.
Only one patient with cryptogenic hepatitis died and had abnormal levels for both Cu and Zn. No patient was transplanted.
Although there are only a few studies on Cu and Zn levels in acute hepatitis, including a limited number of patients with only viral causes13,14,16, the effect of these trace elements on patients with chronic hepatitis or cirrhosis has attracted the attention of many researchers. Kalkan et al16, Grungreiff et al19 and Cesur et al15 calculated Cu and/or Zn levels in a different setting of patients with chronic hepatitis. Kalkan et al16 showed significantly reduced Zn and elevated Cu levels in patients with chronic hepatitis compared to healthy control subjects. Grungreiff et al19 and Cesur et al15 reported similar Zn and Cu concentrations in patients with chronic hepatitis and healthy control subjects. On the other hand, patients with decompensated liver cirrhosis exhibited a more pronounced reduction in Zn concentrations19.
Cu and Zn are absorbed by the small intestine and are bound to plasma albumin. Cuis absorbed by a specific intestinal transport mechanism and is carried to the liver where it is incorporated into the protein transporter ceruloplasmin which delivers Cu to target tissues in the body. In the liver, Cu is stored by the storage protein metallothionein20. Zn after absorption by the small intestine is carried to the liver by portal circulation. Zn absorption is regulated by matallotheionin which binds both Cu and Zn and also acts as a Zn storage protein in the liver21.
Different mechanism of Cu and Zn homeostasis may play a role in acute hepatitis compared to chronic hepatitis or liver cirrhosis. In chronic hepatitis proinflammatory cytokines and particularly increase of IL-6 may play an important role in trace element levels22,23.In liver cirrhosis, the changes in the albumin synthesis, intestinal resorption and use of hepatic zinc are disturbed inducing low zinc levels18.However, Cu concentration in liver cirrhosis is a controversial issue19. Metal binding proteins such as metallothionein or ceruloplasmin are included in the family of acute phase proteins. In acute infections, acute phase proteins are increased and consequently metal binding proteins acting as metal transporters or storage proteins for trace elements are also increased. In the first days of acute infection, the metallothionein expression increases in the liver and intestine of experimental animals10. The increase of metallothionein in the liver was found to correlate positively with both Zn and Cu in previous studies10.Consequently during hepatocyte injury in acute infection, a leak of trace elements to the blood may occur10. In the current study, Zn exhibited positive correlation with ALT which is a marker of hepatocyte damage.
According to the above study10,low or high levels of Zn in acute hepatitis depend on the time elapsing since the onset of infection. Zn levels are elevated in the first days of infection and they are diminished thereafter10. Our results showed a variety of alterations above and below normal values in both Cu and Zn concentrations at the clinical onset of acute hepatitis compared to healthy controls. We do not have sequential sera in order to determine possible alterations of trace elements during the course of the disease. Many factors may account for high and low abnormal trace element values in acute hepatitis, including damage of the hepatocytes – leaking of trace elements and elevated metal binding proteins for the former and reduced intestinal absorption as well as low albumin synthesis for the latter. The controversy between our results and those of Pramoolsinap et al14,Fota-Markowska et al13, or Kalkan et al16,may be attributed to different setting of patients since the above studies included only patients with acute hepatitis B.
Gender-specific concentrations were not found in our study. Zn differences were prominent in both men and women compared to healthy controls while Cu concentrations were practically the same. Gender-specific differences in both Cu and Zn were not found between men and women in the acute hepatitis group but they were evident in the control group. The higher Cu in females of the control group was in agreement with Grungreiff et al19,but this gender-specific difference was not confirmed in our patient group. Hormonal contraception was not used in patients and controls and expected changes induced by sex hormone use19 were not apparent in the current study.
Another finding of the current study was that levels of Cu and Zn were similar irrespective of the etiology of acute hepatitis and the frequency of patients with abnormal values of trace elements did not differ among variable etiologies of acute hepatitis. In a previous study on patients with autoimmune hepatitis Zn levels were described as low24 but the timing of tissue sampling in the above inflammatory conditions was not clear. It was not stated whether Zn and Cu were calculated during acute exacerbations or during a quiescent remission period of autoimmune hepatitis. It is therefore possible that there are elevations and reductions of trace elements concentrations, depending on the phase of the disease. Moreover, we found that in severe cases of acute hepatitis, Cu and Zn exhibited positive correlations with prothrombin time and ALT respectively, reflecting correlations with severity and activity of the disease.
It must be noted that the current study is based on preliminary data. For this reason, no sequential data during the course of the disease are exhibited and the sizes of groups of different etiologies are small.
In conclusion, serum Cu and Zn levels display wide variations in patients with acute hepatitis. Zn abnormalities at the onset of the disease are usually above normal and thus Zn supplementation is not necessary. Cu abnormalities may be above or below normal and more investigation is needed for its role in the severity of the disease.
Conflict of interest
1. Danks DM. Copper deficiency in humans. Annu Rev Nutr. 1988; 8: 235-257.
2. Prasad AS. Zinc: role in immunity, oxidative stress and chronic inflammation. Curr Opin Clin Nutr Metab Care. 2009; 12: 646-652.
3. Berg JM, Shi Y. The galvanization of biology: a growing appreciation for the roles of zinc. Science. 1996; 271: 1081-1085.
4. Zalewski PD, Forbes IJ, Giannakis C. Physiological role for zinc in prevention of apoptosis (gene-directed death). Biochem Int. 1991; 24: 1093-1101.
5. Fung EB, Ritchie LD, Woodhouse LR, Roehl R, King JC. Zinc absorption in women during pregnancy and lactation: a longitudinal study. Am J Clin Nutr. 1997; 66: 80-88.
6. Krebs NF. Overview of zinc absorption and excretion in the human gastrointestinal tract. J Nutr. 2000; 130: 1374S-1377S.
7. Silverio Amancio OM, Alves Chaud DM, Yanaguibashi G, Esteves Hilário MO. Coppr and zinc intake and serum levels in patients with juvenile rheumatoid arthritis. Eur J Clin Nutr. 2003; 57: 706-712.
8. Zowczak M, Iskra M, Paszkowski J, Manczak M, Torlinski L, Wysocka E. Oxidase activity of ceruloplasmin and concentrations of copper and zinc in serum of cancer patients. J Trace Elem Med Biol. 2001; 15: 193-196.
9. Penkowa M, Hidalgo J. Metallothionein I+II expression and their role in experimental autoimmune encephalomyelitis. Glia. 2000; 32: 247-263.
10. Ilbäck NG, Frisk P, Tallkvist J, Gadhasson IL, Blomberg J, Friman G. Gastrointestinal uptake of trace elements are changed during the course of a common human viral (Coxsackievirus B3) infection in mice. J Trace Elem Med Biol. 2008; 22: 120-130.
11. Matousek de Abel de la Cruz AJ, Burguera JL, Burguera M, Añez N. Changes in total content of iron, copper and zinc in serum, heart, liver, spleen and skeletal muscle tissues of rats infected with Trypanosoma cruzi. Biol Trace Elem Res. 1993; 37: 51-70.
12. Overbeck S, Rink L, Haase H. Modulating the immune response by oral zinc supplementation: a single approach for multiple diseases. Arch Immunol Ther Exp (Warsz). 2008; 56: 15-30.
13. Fota-Markowska H, Przybyla A, Borowicz I, Modzewska R. Serum zinc (Zn) level dynamics in blood serum of patients with acute viral hepatitis B and early recovery period. Ann Univ Mariae Curie Sklodowska Med. 2002; 57: 201-209.
14. Pramoolsinsap C, Promvanit N, Kurathong S. Serum trace metal levels in patients with acute hepatitis B. Southeast Asian J Trop Med Public Health. 1996; 27: 476-480.
15. Cesur S, Cebeci SA, Kavas GO, Aksaray S, Tezeren D. Serum copper and zinc concentrations in patients with chronic hepatitis B. J Infect. 2005; 51: 38-40.
16. Kalkan A, Bulut V, Avci S, Celik I, Bingol NK. Trace elements in viral hepatitis. J Trace Elem Med Biol. 2002; 16: 227-230.
17. Kouremenou-Dona E, Dona A, Papoutsis J, Spiliopoulou C. Copper and zinc concentrations in serum of healthy Greek adults. Sci Total Environ. 2006; 359: 76-81.
18. Hennes EM, Zeniya M, Czaja AJ, Parés A, Dalekos GN, Krawitt EL, et al; International Autoimmune Hepatitis Group. Simplified criteria for the diagnosis of autoimmune hepatitis. Hepatology. 2008; 48: 169-176.
19. Grüngreiff K, Hebell T, Gutensohn K, Reinhold A, Reinhold D. Plasma concentrations of zinc, copper, interleukin-6 and interferon-?, and plasma dipeptidyl peptidase IV activity in chronic hepatitis C. Mol Med Rep. 2009; 2: 63-68.
20. Harris ED. The iron-copper connection: the link to ceruloplasmin grows stronger. Nutr Rev. 1995; 53: 170-173.
21. Sandström B. Bioavailability of zinc. Eur J Clin Nutr. 1997; 51 Suppl 1: S17-S19.
22. Grüngreiff K, Reinhold D, Ansorge S. Serum concentrations of sIL-2R, IL-6, TGF-beta1, neopterin, and zinc in chronic hepatitis C patients treated with interferon-alpha. Cytokine. 1999; 11: 1076-1080.
23. Neuman MG, Benhamou JP, Malkiewicz IM, Akremi R, Shear NH, Asselah T, et al. Cytokines as predictors for sustained response and as markers for immunomodulation in patients with chronic hepatitis C. Clin Biochem. 2001; 34: 173-182.
24. Pereira TC, Saron ML, Carvalho WA, Vilela MM, Hoehr NF, Hessel G. Research on zinc blood levels and nutritional status in adolescents with autoimmune hepatitis. Arq Gastroenterol. 2011; 48: 62-65.