Ceruloplasmin levels in human sera from various diseases and their correlation with patient’s age and gender

Viorica Lopez-Avila, William H. Robinson, Kirk Lokits

.

DOI: 10.4236/health.2009.12017                

Abstract

Ceruloplasmin (Cp), a copper metalloprotein in human serum has been a valuable diagnostic marker in Wilson’s disease where Cp levels tend to be low while high levels in serum were asso-ciated with myocardial infarction, neoplastic and inflammatory conditions. There is no stan-dardized reference method for Cp and current immunologic and bichromatic assays have a number of drawbacks. The method described here uses immunoaffinity chromatography to remove six of the most abundant proteins from a serum sample and high-pressure liquid chro- matography (HPLC) with a size-exclusion col-umn to separate Cp from other serum proteins and any free Cu prior to analysis of 63Cu and 65Cu by inductively-coupled plasma mass spec-trometry (ICPMS). Identification of Cp is based on retention time match of the unknown protein in the serum sample with the Cp external stan-dard and the presence of 63Cu and 65Cu at a ratio of 2.2 ± 0.1. The method accuracy, as estab-lished independently by two of the authors with a reference serum certified for Cp, is 98 to 101% and the coefficient of variation is 6.4% and 5.4%, respectively. The assay was used to analyze a total of 167 human sera for Cp from patients with myocardial infarction (MI), pulmonary em-bolism (PE), rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), other forms of ar-thritis, and a set of healthy patients as normal controls (NC). Our data show that Cp concen-trations tend to be higher in MI, RA, and SLE patients, higher in female as compared to male patients, and we did not observe a correlation between Cp concentration and patient’s age for the set of 70 patients for which we had gender and age information.

Keywords

Ceruloplasmin; HPLC-ICPMS; Immu-noaffinity Chromatography

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	Takahashi, N., Ortel, T., and Putnam, F. (1984) Sin-gle-chain structure of human ceruloplasmin: The com-plete amino acid sequence of the whole molecule. Proc Nat Acad Sci USA, 81, 390-4.
[2]	Hellman, N.E. and Gitlin, J.D. (2002) Ceruloplasmin metabolism and function. Annu Rev Nutr, 22, 439-458.
[3]	Hellman, N.E., Kono, S., Mancini, G.M., Hoogeboom, A.J., de Jong, G.J., and Gitlin, J.D. (2002) Mechanisms of copper incorporation into human ceruloplasmin. J Biol Chem, 48, 46632-46638.
[4]	Linder, M.C., Wooten, L., Cerveza, P., Cotton, S., Shulze, R., and Lomeli, N. (1998) Copper transport. Am J Clin Nutr, 67, 965S-971S.
[5]	Prohanska, J.R. and Gybina, A.A., (2004) Intracellular copper transport in mammals. J Nutr, 134, 1003-1006.
[6]	DakoCytomation (1998) Application note guideline for determination of ceruloplasmin in serum/plasma on hi-tachi 911, 3.
[7]	Buffone, G.J., Brett, E.M., Lewis, S.A., Iosefson, M., and Hicks, J.M. (1979) Limitations of immunochemical measurement of ceruloplasmin. Clin Chem, 25, 749-51.
[8]	Hohbadel, D.C., McNeely, M.D., and Sunderman, F.W. (1975) Automated bichromatic analysis of serum ceru-loplasmin. Annals Clin Lab Sci, 5, 65-70.
[9]	Twomey, P.J., Viljoen, A., House, I.M., Reynolds, T.M., Wierzbicki, A.S., (2005) Relationship between serum copper, ceruloplasmin, and non-ceruloplasmin –bound Copper in routine clinical practice. Clin Chem, 51, 1558-9.
[10]	Macintyre, G., Gutfreund, K.S., Martin, W.R., Camicioli, R., Cox, D.W. (2004) Value of an enzymatic assay for the determination of serum ceruloplasmin. J. Lab. Clin. Med. 144, 294-301.
[11]	Hahn, S.-H., Jang, Y.-J., Lee, S.-Y., Shin, H.-C., Park, S.-Y., Yu, E.-S., and Han, H.-S. (2004) US patent 6,806,044 B2–Method of measuring ceruloplasmin con-centration in a blood spot, kit and method of diagnosing Wilson’s disease.
[12]	Newsome, D.A., Swartz, M., Leone, N.C., Hewitt, A.T., Wolford, F., and Miller, E.D. (1986) Macular degenera-tion and elevated serum ceruloplasmin. Investig Ophtal-mol &Visual Sci, 27, 1675-80.
[13]	Reunanen, A., Knekt, P., and Aaran, R.K. (1992) Serum ceruloplasmin level and the risk of myocardial infarction and stroke. Amer J Epidemiol, 136, 1082-90.
[14]	Interpath Laboratory, Inc. www.interpathlab.com/TechnicalUpdates/ceruloplasmin.htm (Accessed November 2006)
[15]	Lopez-Avila, V., Robinson, W.H., and Sharpe, O. (2006) Determination of ceruloplasmin in human serum by SEC- ICPMS. Anal Bioanal Chem, 386, 180-7.
[16]	Emons, H. (2005) Certificate of analysis ERM DA470, Institute for Reference Materials and Measurements, Geel, Belgium.
[17]	Baudner, S., Bienvenu, J., Blirup-Jensen, S., Calstrom, A., Johnson, A.M., Milford Ward, A., et al. (2005) Certifica-tion report – The certification of a matrix reference mate-rial for immunochemical measurement of 15 serum pro-teins, institute for reference materials and measurements, Geel, Belgium.
[18]	Hanzschel, H., Bird, H.A., Seidel, W., Kruger, W., Neu-mann, G., Schneider, G., and Wright, V. (1991) Polym-yalgia rheumatica and rheumatoid arthritis of the elderly: A clinical, laboratory, and scintigraphic comparison. An-nals of the Rheumatic Diseases, 50, 619-22.
[19]	Lyngbye, J. and Kroll, J. (1971) Quantitative immu-noelectrophoresis of protein in serum from a normal population: season-, age-, and sex-related variations. Clin Chem, 17, 495-500.
[20]	Sontakke, A.N. and More, U. (2004) Changes in serum ceruloplasmin levels with commonly used methods of contraception. Indian J Clin Biochem, 19(1), 102-104.
[21]	Revnic, F. (1995) The significance of serum ceruloplas-min in diagnosis of rheumatoid arthritis, Toxicol Lett, 78 (1), 70-71.
[22]	Musci, G., Bonaccorsi, di Patti, M.C., Fagiolo, U., and Calabrese, L. (1993) Age-related changes in human ce-ruloplasmin, J Biol Chem, 268 (18), 13388-13395.