cant elevations of serum copper and ceruloplasmin levels in lymphoma patients and ... eral health, medication usage, and other variables that might influence ...
Elevations in Serum Copper, Erythrocytic Copper, and Ceruloplasmin Concentrations in Smokers GARY N. DAVIDOFF, B.S., MAY L. VOTAW, M.D., WILLIAM W. COON, M.D., DONALD E. HULTQUIST, PH.D., BRIAN J. FILTER, B.S., AND STEPHEN A. WEXLER
Davidoff, Gary N., Votaw, May L., Coon, William W., Hultquist, Donald E., Filter, Brian J., and Wexler, Stephen A.: Elevations in serum copper, erythrocytic copper, and ceruloplasmin concentrations in smokers. Am J Clin Pathol 70: 790-792, 1978. Previous investigators have reported significant elevations of serum copper and ceruloplasmin levels in lymphoma patients and subjects using estrogen, but have not taken into account the smoking habits of these populations. In order to determine whether smoking had any effect on these variables, the authors examined five groups for serum and erythrocytic copper and ceruloplasmin levels: 40 healthy subjects, 14 users of oral contraceptives, 25 smokers, 14 nonsmoking pretreatment lymphoma patients, and eight treated lymphoma patients. Significant elevations of serum copper and ceruloplasmin in pretreatment lymphoma patients, treated patients, and estrogen users were found, confirming previous reports. In addition, all groups had elevations of erythrocytic copper compared with the healthy subjects. Also, significant elevations of serum copper (P < .0053) and ceruloplasmin (P < .0001) were found in smoking relative to nonsmoking subjects. No correlation between duration of smoking and these elevations was found. (Key words: Smoking; Serum copper; Erythrocytic copper; Ceruloplasmin; Lymphoma; Estrogen.) PREVIOUS INVESTIGATORS have reported variations from normal in serum copper and/or ceruloplasmin levels in Hodgkin's and non-Hodgkin's lymphoma, 515 Wilson's disease, 3,9 myocardial infarction, 16 and sicklecell anemia. 10 Elevations from normal of these two modalities have also been found in users of oral conHowever, none of these studies traceptives. differentiated between smoking and nonsmoking subjects. This paper presents the levels of serum and erythrocytic copper and ceruloplasmin in smoking versus nonsmoking subjects and discusses the clinical significance. Materials and Methods We examined specimens from 97 subjects: 40 healthy subjects who were nonsmokers, four users of oral con-
University of Michigan Medical Center and the School of Public Health Laboratories, Ann Arbor, Michigan
traceptives, 25 smokers, four nonsmoking pretreatment lymphoma patients, and eight lymphoma patients during treatment. Subjects were selected from healthy students and staff volunteers at the University of Michigan and patients at University Hospital, Ann Arbor, Michigan. Demographic data are listed in Table 1. After giving informed consent, each subject was given a comprehensive questionnaire concerning diet, general health, medication usage, and other variables that might influence copper and/or ceruloplasmin levels. All sampling was done during the early morning office hours after a light breakfast (e.g., orange juice and 1 ounce of cereal) or after an eight-hour fast. Samples of blood were drawn by venipuncture into 15-ml VacU-Tainers* that were free of additives or preservatives. Samples were centrifuged within three hours; the serum was decanted and frozen in small plastic vials until analyzed. The erythrocytic clots remained in the sampling tubes at 4 C until assay. All plasticware and glassware was scrubbed rigorously with Alconoxt solution, rinsed three times in distilled water, leached for 24 hours in 50% nitric acid, analytic grade,$ rinsed three times in distilled water, and dried in a sterile oven. Serum copper analysis was done using a Varian§ A120 flame atomic absorption spectrometer operating at 324.8 nm with an air-acetylene flame. The serum samples were diluted 10:1 in replicate pairs with 0 . 1 5 M NaCl solution (analytic grade). Quality controls for precision and recovery were employed throughout the investigation. Replicate analysis of 60 samples of plasma from outdated whole blood gave a standard deviation of 2%. Serum recovery was performed by standard addition of portions of 0.1, 0.2, and 0.4 pig/ml copper standard to 20 replicate samples of serum and
Received June 22, 1977; received revised manuscript September 21, 1977; accepted for publication September 21, 1977. * Scientific Products, 1430 Waukegan Road, McGraw Park, Illinois. Supported in part by American Cancer Society Institutional t Alconox Inc., 215 Park Ave., New York, New York. Research Grant IN-40Q. t J. T. Baker Co., Phillipsburg, New Jersey. Address reprint requests to Dr. Votaw: Department of Internal § Varian Associates, 611 Hansen Way, Palo Alto, California. Medicine, Box 21, 160A, College of Medicine, East Tennessee State University, Johnson City, Tennessee 37601. 0002-9173/78/1100/0790 $00.65 © American Society of Clinical Pathologists
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791
COPPER AND CERULOPLASMIN IN SMOKERS
Vol. 70 • No. 5
Table I. Demographic Data on Subjects Sex
Age
Group
Male
Female
Range
Median
Total No. Subjects
Healthy nonsmoking subjects Users of oral contraceptives Smokers Pretreatment patients (10 Hodgkin's disease, 4 non-Hodgkin's lymphoma) Treated patients (4 irradiation, 4 chemotherapy)
25 0 12
15 14 13
18-59 20-39 20-64
25 23 33
40 14 25
5
9
18-77
24
14
3
5
18-54
24
8*
* Includes four patients in the pretreatment patient group.
diluent, as described above. Recoveries for the sample specimens were 93:5%, 96.3%, and 97.0%, respectively. Erythrocytic copper was analyzed with the use of flame atomic absorption spectroscopy, as described above. The erythrocytic clots were weighed and digested with the use of a nitric perchlorate solution (4:1 concentrated acid: 2 ml/g of clot). The ash was redissolved in 3 ml of concentrated hydrochloric acid, diluted to 25 ml in volumetric flasks, and analyzed. The results of 30 ashed samples from the same unit of outdated whole blood gave a standard deviation of 2%. Recovery was performed by standard addition of known amounts of copper to the erythrocytic clot prior to ashing. Ceruloplasmin was analyzed in replicate with the use of the PPD-oxidase method (oxidation of p-phenylene diamine dihydrochloride) of Ravin.12 Replicate analysis of eight samples from each of four
normal subjects each gave a standard deviation of 5%. All analytic and categorical variables were analyzed on an IBM 360 computer using the Michigan Interactive Data Analysis System (MIDAS). Basic descriptive functions, histograms, linear and multiple linear regression, and scatter plots were employed. Results Table 2 lists the sample sizes, means, and two-standard-deviation limits for serum copper, erythrocytic copper, and ceruloplasmin activity for all five groups. Student's t tests demonstrated that the elevations of serum copper, erythrocytic copper, and ceruloplasmin in all four groups compared with healthy nonsmoking subjects were significant at least at the P < 0.01 level for all except erythrocytic copper in smokers,/" < 0.05.
Table 2. Descriptive Statistics
Healthy nonsmoking subjects SCu* Cpt ErCut Users of oral contraceptives SCu
cErCu P Smokers SCu Cp ErCu Pretreatment patients SCu Cp ErCu Treated patients SCu Cp ErCu * SCu " serum copper, /ig/ml. t Cp « ceruloplasmin, mg/100 ml. t ErCu i= erythrocytic copper, /ig/g.
Significance (Student's t Test of Mean Value vs. Normal)
No.
Minimum
Maximum
Mean
2 SD
40 35 24
0.62 18.0 0.48
1.91 45.2 0.85
0.99 30.3 0.65
0.44 12.4 0.16
11 12 5
1.01 30.0 0.67
2.11 61.7 0.84
1.75 52.5 0.77
0.64 16.8 0.13
P < 0.0001 P < 0.0001 P < 0.0090
25 21 12
0.71 20.4 0.47
1.60 58.6 1.32
1.16 41.1 0.77
0.46 22.1 0.49
P < 0.0053 P < 0.0001 P < 0.0438
11 12 6
1.29 26.7 0.70
2.88 70.9 1.41
1.97 52.5 1.03
1.12 25.8 0.47
P < 0.0001 P < 0.0001 P < 0.0001
49 41 38
0.88 26.7 0.53
2.88 83.3 1.41
1.43 46.9 0.78
0.78 27.0 0.35
P < 0.0001 P < 0.0001 P < 0.0013
DAVID0FFE7-AZ..
792
Table 3. Correlation Coefficients between Serum Copper and Ceruloplasmin Group
Regression Line
Healthy nonsmoking subjects Users of oral rcontraceptives nntrarpntivps Smokers Pretreatment patients Treated patients
SCut = 0.39 + 0.02 (Cpt)
0.58
S C u = 0.01 + 0.03 ( C p )
0.87
SCu = 0.94 + 0.01 (Cp) SCu = -0.08 + 0.04 (Cp) SCu = 0.24 + 0.03 (Cp)
NS 0.82 0.85
* r = Pearson correlation coefficient (NS •• not significant). t SCu = serum copper, ^tg/ml. t Cp = ceruloplasmin, mg/100 ml.
Significant correlation coefficients of the linear relationship between serum copper and ceruloplasmin were observed for every population except smokers (Table 3). No correlation was observed between duration of smoking (pack-years smoked), or frequency of usage (packs per day) and elevations of serum copper, erythrocytic copper, or ceruloplasmin. Discussion We have shown that serum copper, erythrocytic copper, and ceruloplasmin activity are elevated from normal levels in lymphoma patients, smokers, and users of oral contraceptives. This work confirms previous studies concerning the elevations from normal of serum copper and ceruloplasmin in lymphoma patients515 and estrogen users.'•4-7i8,u To our knowledge, it is the first report concerning erythrocytic copper levels in smokers, lymphoma patients, or users of oral contraceptives. Also, the significant elevations in smokers relative to nonsmokers in serum copper (P < 0.0053) and ceruloplasmin {P < 0.0001) have not previously been reported. The linear correlation coefficients for the scatter plots of serum copper and ceruloplasmin support previous studies of estrogen users7 and lymphoma patients.14 However, no significant linear correlation was found in the smoking group. The elevations of copper and ceruloplasmin levels in smoking versus nonsmoking populations may have some clinical significance with respect to the methods presently used for monitoring disease activity in lymphoma. Several studies have reported the usefulness of serial serum copper or ceruloplasmin determinations as an indirect measure of disease activity in Hodgkin's and non-Hodgkin's lymphoma.2-613 These investigators reported dramatic decreases in serum copper and ceruloplasmin levels with successful treatment. When
A.J.C.P. • November 1978
serum copper or ceruloplasmin levels, or both, are used to assess the activity of lymphoma, the need to ascertain whether the patient smokes is very important. It is conceivable that patients who stop smoking in the middle of their treatment may develop serum copper/ ceruloplasmin curves indicative of remission. Conversely, individuals who begin to smoke after total remission may have serum copper/ceruloplasmin levels indicative of relapse. Acknowledgments. The following individuals contributed valuable technical assistance: Marc Feldman, Jerome Finkel, Lawrence Handler, Gary Hoberman, Robert Huler, Paul Juengel, Edward Levin, Jon Ross, Sam Shamie, Kenneth Shapiro, Jeffrey Weitz, Bennett Yu, and Barry Zisholtz.
References 1. Carruthers ME, Hobbs CB, Warren RL: Raised serum copper and caeruloplasmin levels in subjects taking oral contraceptives. J Clin Pathol 19:498-450, 1966 2. Davidoff GN, Votaw ML, Hecker LH, et al: Serum copper and ceruloplasmin levels as an indicator of disease activity of lymphoma. Clin Res 25:337F, 1977 3. Evans GW: Copper hemostasis in the mammalian system. Physiol Rev 53:535-570, 1973 4. Hambidge KM, Droegemuller W: Changes in plasma and hair concentrations of zinc, copper, chromium, and manganese during pregnancy. Obstet Gynecol 44:666-672, 1974 5. Hrgovcic M, Tessmer CF, Mumford DM, et al: Interpreting serum copper levels in Hodgkin's disease. Tex Med 71: 53-63, 1975 6. Hrgovcic M, Tessmer CF, Thomas FB, et al: Significance of serum copper levels in adult patients with Hodgkin's disease. Cancer 31:1337-1345, 1973 7. Margen S, King JC: Effects of oral contraceptive agents on the metabolism of some trace minerals. Am J Clin Nutr 28: 392-402, 1975 8. Musa BU, Doc RP, Seal US: Serum protein alterations produced in women by synthetic estrogens. J Clin Endocrinol 27:1403-1469, 1967 9. Neumann PZ, Silverberg M: Metabolic pathways of red blood cell copper in normal humans and in Wilson's disease. Nature 213:775-779, 1967 10. Olatumbosum DA, Isaacs-Sodeye WA, Adeniyi FA, et al: Serum-copper in sickle-cell anaemia-Letter. Lancet 1:7901, 1975 11. Prasad AS: Effects of oral contraceptives on nutrients. II. Minerals. Am J Clin Nutr 28:377-384, 1975 12. Ravin HA: An improved colorimetric enzymatic assay of ceruloplasmin. J Lab Clin Med 58:161-168, 1961 13. Tessmer DC, Hrgovcic M, Thomas FB, et al: Serum copper as an index of tumor response to radiotherapy. Radiology 106:635-639, 1973 14. Thorling EB, Jensen KB, Andersen CJ, et al: Cobre serico y ceruloplasmina en la enfermed de Hodgkin. Folia Clin Int 15:2-12, 1965 15. Thorling EB, Thorling K: The clinical usefulness of serum copper determinations in Hodgkin's disease. Cancer 38: 225-231, 1976 16. Varsieck J, Barbier F, Speecke A: Influence of myocardial infarction on serum manganese, copper, and zinc concentrations. Clin Chem 21:578-581, 1975
