Biological Trace Element Research

, Volume 130, Issue 3, pp 229–240 | Cite as

Relevance of Non-ceruloplasmin Copper to Oxidative Stress in Patients with Hepatocellular Carcinoma

  • Arumugam GeethaEmail author
  • Panneerselvam Saranya
  • Sam Annie Jeyachristy
  • Rajagopal Surendran
  • Arunachalam Sundaram


Altered copper homeostasis and oxidative stress have been observed in patients with hepatocellular carcinoma. Non-ceruloplasmin copper, the free form, is a potent pro-oxidant than the protein bound copper. The aim of the present study was to evaluate which form of copper can be correlated with the oxidative stress in the circulation and in the malignant liver tissues of hepatocellular carcinoma patients. Hepatocellular carcinoma patients (grades II and III, n = 18) were enrolled in this study. Serum levels of total, free and bound copper, ceruloplasmin, iron, iron-binding capacity, lipid peroxidation products, and enzymatic and non-enzymatic antioxidants were quantified in serum and in malignant liver tissues and compared with those of normal samples (n = 20). A significant positive correlation between the serum non-ceruloplasmin copper and lipid peroxidation products and negative correlation with antioxidants were observed in hepatocellular carcinoma patients. In liver tissue, glutathione peroxidase, superoxide dismutase, and catalase activity were significantly decreased with concomitant elevation in oxidative stress markers. Our experiment revealed that the elevation in non-ceruloplasmin copper has high relevance with the oxidative stress than the bound copper.


Hepatocellular carcinoma Ceruloplasmin Non-ceruloplasmin copper Oxidative stress Antioxidants 


  1. 1.
    Clark HP, Carson WF, Kavanagh PV, Ho CP, Shen (2005). Staging and current treatment of hepatocellular carcinoma. Radiographics. 25Suppl 1:S3–23 (Review: S3–23).PubMedCrossRefGoogle Scholar
  2. 2.
    EI-Serag HB, Mason AC (1999). Rising incidence of hepatocellular carcinoma in the United States. N Engl J Med. 340: 745–509.CrossRefGoogle Scholar
  3. 3.
    Chopra P, Vijayaragvan M, Nayak NC (1987). Significance of liver cell dysplasia in cirrhosis and hepatocellular carcinoma. Indian J Med Res. 6:382–90.Google Scholar
  4. 4.
    Sheila Sherlock and James Dooley (2002). Diseases of the liver and biliary system. Eleventh Edition 2002 by Blackwell Science LTD a Blackwell Publishing.Google Scholar
  5. 5.
    Wiseman H, Halliwell B (1996). Damage to DNA by reactive oxygen and nitrogen species: role in inflammatory disease and progression to cancer. Biochem J. 313: 7–29.Google Scholar
  6. 6.
    Farinati F, Cardin R, Bortolami M, Burra P, Russo FP (2007). Hepatitis C virus: from oxygen free radicals to hepatocellular carcinoma. J Viral Hepatitis. 4: 21–99.Google Scholar
  7. 7.
    Yang LY, Chen WL, Lin JW, Lee SF, Lee CC (2005). Differential expression of antioxidant enzymes in various hepatocellular carcinoma cell lines. J Cell Biochem. 6: 622–631.CrossRefGoogle Scholar
  8. 8.
    Halliwell BM, Gutterige JMC (2007). Free radicals in biology and medicine 4th Edition. Oxford University Press, New York.Google Scholar
  9. 9.
    Kubo S, Fukuda H, Ebara M, Ikota N, Saisho H (2005). Evaluation of distribution patterns for copper and zinc in metallothionein and superoxide dismutase in chronic liver diseases and hepatocellular carcinoma using high-performance liquid chromatography (HPLC). Biol Pharm Bull. 28: 1137–41.PubMedCrossRefGoogle Scholar
  10. 10.
    Hu GF (1998). Copper stimulates proliferation of human endothelial cells under culture. J Cell Biochem. 69: 326–35PubMedCrossRefGoogle Scholar
  11. 11.
    Valko M, Morris H, Cronin MT (2005). Metals, toxicity and oxidative stress. Curr Med Chem. 12: 1161–1208.PubMedCrossRefGoogle Scholar
  12. 12.
    Evenson MA, Warren BL (1975). Determination of serum copper by atomic absorption, with use of the graphite cuvette. Clin Chem. 21: 619–25.PubMedGoogle Scholar
  13. 13.
    Carthew GW, Dey RL (1985). A rapid tissue extraction method for determining liver copper content by atomic absorption spectroscopy. N Z Vet J. 33: 168–70.PubMedGoogle Scholar
  14. 14.
    Ramsay WN (1953). The determination of iron in blood plasma or serum. Biochem J. 53: 227–31.PubMedGoogle Scholar
  15. 15.
    Sunderman FW, Nomoto S (1970). Measurement of human serum ceruloplasmin by its p-phenylenediamine oxidase activity. Clin Chem. 16: 903–10.PubMedGoogle Scholar
  16. 16.
    Walshe JM (2003). Wilson’s disease: the importance of measuring serum ceruloplasmin non-immunologically. Ann Clin Biochem. 40: 115–21.PubMedCrossRefGoogle Scholar
  17. 17.
    Gaffney D, Fell GS, O’Reilly DS (2000). ACP Best Practice no. 163. Wilson’s disease: acute and presymptomatic laboratory diagnosis and monitoring. J Clin Pathol. 53: 807–812.PubMedCrossRefGoogle Scholar
  18. 18.
    Ramsay WN (1954). An improved technique for the determination of plasma iron. Biochem. J: 57 (328th meeting): xvii.Google Scholar
  19. 19.
    Draper HH, Hadley M (1990). Malondialdehyde determination as index of lipid peroxidation. Methods Enzymol. 46: 421–31.CrossRefGoogle Scholar
  20. 20.
    Jiang ZY, Hunt JV, Wolff SP (1992). Ferrous ion oxidation in the presence of xylenol orange for detection of lipid hydroperoxide in low density lipoprotein. Anal Biochem. 202: 384–9.PubMedCrossRefGoogle Scholar
  21. 21.
    Miranda KM, Espey MG, Wink DA (2001). A rapid, simple spectrophotometric method for simultaneous detection of nitrate and nitrite. Nitric oxide. 5: 62–71.PubMedCrossRefGoogle Scholar
  22. 22.
    Cook JA, Kim SY, Teague D, Krishna MC, Pacelli R (1996). Convenient colorimetric and fluorometric assays for S-nitrosothiols. Anal Biochem. 238: 150–8.PubMedCrossRefGoogle Scholar
  23. 23.
    Khan J, Brennand DM, Bradley N, Gao B, Bruckdorfer R (1998). 3-Nitrotyrosine in the proteins of human plasma determined by an ELISA method. Biochem J. 330: 95–801.Google Scholar
  24. 24.
    Kakkar P, Das B, Viswanathan PN (1984). A modified spectrophotometric assay of superoxide dismutase. Indian J Biochem Biophys. 21: 30–2.Google Scholar
  25. 25.
    Sinha AK (1972). Colorimetric assay of catalase. Anal Biochem. 47, 89–94.CrossRefGoogle Scholar
  26. 26.
    Flohe L, Gunzler WA (1984). Assays of glutathione peroxidase. Methods Enzymol 5: 114–21.CrossRefGoogle Scholar
  27. 27.
    Misra HP, Fridovich I (1977). Superoxide dismutase: “positive” spectro-photometric assays. Anal Biochem. 79: 553–60.PubMedCrossRefGoogle Scholar
  28. 28.
    Moron MS, Depierre JW, Mannervik B (1979). Levels of glutathione, glutathione reductase and glutathione S-transferase activities in rat lung and liver. Biochim Biophys Acta. 582: 67–78.PubMedGoogle Scholar
  29. 29.
    Baker H, Frank O, De Angelis B, Feingold S (1980). Plasma tocopherol in man at various times after ingesting free or acetylated tocopherol. Nutr Rep Int. 21:531–6.Google Scholar
  30. 30.
    Jacob RA (1990). Assessment of human vitamin C status. J Nutr. 120: 1480–5.PubMedGoogle Scholar
  31. 31.
    Bradford MM (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 72: 248–54.PubMedCrossRefGoogle Scholar
  32. 32.
    Poo JL, Rosas-Romero R, Montemayar AC, Isoard F, Uribe M (2003). Diagnostic value of the copper/zinc ratio in hepatocellular carcinoma: a case control study. J Gastroenterol. 38: 45–51.PubMedCrossRefGoogle Scholar
  33. 33.
    Lowndes SA, Harris AL (2005). The role of copper in tumor angiogenesis. J Mammary Gland Biol Neoplasia. 10: 299–310.PubMedCrossRefGoogle Scholar
  34. 34.
    Wu T, Sempos CT, Freudenheim JL, Muti P, Smit E (2004). Serum iron, copper and zinc concentrations and risk of cancer mortality in US adults. Ann Epidemiol. 14: 195–201.PubMedCrossRefGoogle Scholar
  35. 35.
    Goodman VL, Brewer GJ, Merajver SD (2004). Copper deficiency as an anti-cancer strategy. Endocr Relat Cancer. 11: 255–63.PubMedCrossRefGoogle Scholar
  36. 36.
    Brewer GJ (2003). Copper-lowering therapy with tetrathiomolybdate for cancer and diseases of fibrosis and inflammation. J Trace Elements and Exp Med. 164: 191–99.CrossRefGoogle Scholar
  37. 37.
    Oqihara H, Oqihara T, Miki M, Yasuda H, Mino M (1995). Plasma copper and antioxidant status in Wilson’s disease. Pediatr Res. 37: 219–26.Google Scholar
  38. 38.
    Geetha A, Jeyachristy SA, Selvamathy SM, Ilavarasi S, Surendran R (2007). A study on the concentrations of serum zinc, non-ceruloplasmin copper, reactive oxygen and nitrogen species in children with Wilson’s disease. Clin Chim Acta. 383: 165–7.PubMedCrossRefGoogle Scholar
  39. 39.
    Babiloni C, Squitti R, Del Percio C, Cassetta E, Ventriqlia MC (2007). Free copper and resting temporal EEG rhythms correlate across healthy, mild cognitive impairment, and Alzheimer’s disease subjects. Clin Neurophysiol. 118: 1244–60.PubMedCrossRefGoogle Scholar
  40. 40.
    Das D, Tapryal N, Goswami SK, Fox PL, Mukhopadhvay CK (2007). Regulation of ceruloplasmin in human hepatic cells by redox active copper: identification of a novel AP-1 site in the ceruloplasmin gene. Biochem J. 402 1: 135–41.PubMedCrossRefGoogle Scholar
  41. 41.
    Battisti C, Formichi P, Tripodi SA, Vindigni C, Roviello F (2000). Vitamin E serum levels and gastric cancer: results from a cohort of patients in Tuscany, Italy. Cancer Lett. 151(1): 15–8.PubMedCrossRefGoogle Scholar
  42. 42.
    Ohshima H, Bartsch H (1994). Chronic infections and inflammatory processes as cancer risk factors: possible role of nitric oxide in carcinogenesis. Mutat Res. 305(2): 253–64.PubMedGoogle Scholar
  43. 43.
    Wang YZ, Cao YQ, Wu JN, Chen M, Cha XY (2005). Expression of nitric oxide synthase in human gastric carcinoma and its relation to p53, PCNA. World J Gastroenterol. 11: 46–50.PubMedGoogle Scholar
  44. 44.
    Wink DA, Hanbauer I, Grisham MB, Laval F, Nims RW (1996). Chemical biology of nitric oxide: regulation and protective and toxic mechanisms. Curr Top Cell Regul. 34: 159–87.PubMedCrossRefGoogle Scholar
  45. 45.
    Shiva S, Wang X, Ringwood LA, Xu X, Yuditskaya S (2006). Ceruloplasmin is a NO oxidase and nitrite synthase that determines endocrine NO homeostasis. Nat Chem Biol. 2: 486–93.PubMedCrossRefGoogle Scholar
  46. 46.
    Kooy NW, Royall JA, Ye YZ, Kelly DR, Beckman JS (1995). Evidence for in vivo peroxynitrite production in human acute lung injury. Am J Respir Crit Care Med. 151: 1250–4.PubMedGoogle Scholar
  47. 47.
    Haddad IY, Pataki G, Hu P, Galliani C, Beckman JS, (1994). Quantitation of nitrotyrosine levels in lung sections of patients and animals with acute lung injury. J Clin Invest. 94: 2407–13.PubMedCrossRefGoogle Scholar
  48. 48.
    Rennenberg H (1982). Glutathione metabolism and possible roles in higher plants. Phytochem. 21: 2771–2781.CrossRefGoogle Scholar
  49. 49.
    Hultberg M, Isaksson A, Andersson A, Hultberg B (2007). Traces of copper ions deplete glutathione in human hepatoma cell cultures with low cysteine content. Chem Biol Interact. 167: 56–62.PubMedCrossRefGoogle Scholar
  50. 50.
    Estrela Jm, Ortega A, Obrador E (2006). Glutathione in cancer biology and therapy. Crit Rev Clin Lab Sci. 43: 143–81.PubMedCrossRefGoogle Scholar
  51. 51.
    Kawamura T, Ohisa Y, Abe Y, Ishimori A, Shineha R (1992). Plasma lipid peroxides in the operation of esophageal cancer. Rinsho Byori. 40: 881–4.PubMedGoogle Scholar
  52. 52.
    Yasuda M, Takesue F, Inutsuka S, Honda M, Nozoe T (2002). Prognostic significance of serum superoxide dismutase activity in patients with gastric cancer. Gastric Cancer. 5: 148–153.PubMedCrossRefGoogle Scholar
  53. 53.
    Izutani R, Asano S, Imano M, Kuroda D, Katao M (1998). Expression of manganese superoxide dismutase in esophageal and gastric cancers. J Gastroenterol. 33: 816–822.PubMedCrossRefGoogle Scholar
  54. 54.
    Inoue M, Sato EF, Nishikawa M, Park AM, Kira Y (2003). Mitochondrial generation of reactive oxygen species and its role in aerobic life. Curr Med Chem. 10: 2495–2505.PubMedCrossRefGoogle Scholar
  55. 55.
    Uhlig S, Wendel A (1992). The physiological consequences of glutathione variations. Life Sci. 51: 1083–94.PubMedCrossRefGoogle Scholar
  56. 56.
    Xu D, Du W, Zhao L, Davey AK, Wang J (2008). The neuroprotective effects of isosteviol against focal cerebral ischemia injury induced by middle cerebral artery occlusion in rats. Planta Med. 74: 816–21.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2009

Authors and Affiliations

  • Arumugam Geetha
    • 1
    Email author
  • Panneerselvam Saranya
    • 1
  • Sam Annie Jeyachristy
    • 1
  • Rajagopal Surendran
    • 2
  • Arunachalam Sundaram
    • 3
  1. 1.Department of BiochemistryBharathi Women’s CollegeChennaiIndia
  2. 2.Department of Surgical Gastroenterology and ProctologyStanley Medical College and HospitalChennaiIndia
  3. 3.Department of PathologyStanley Medical College and HospitalChennaiIndia

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