Measurement of Serum Ferritin by Radioimmunoassay: Results in Normal Individuals and Patients With Breast Cancer

1,2

Donald M. Marcus and Nira Zinberg

3, 4

791-795, 1975.

Ferritins are a family of isomeric proteins (isoferritins) that have an important function in iron storage and metabolism (1, 2). The concentration of ferritin is high· est in liver, spleen, and bone marrow, but these proteins are probably present in every mammalian tissue (1, 2). Elevated levels of ferritin in many tumors and the sera of tumor.bearing individuals have stimulated interest in ferritin as a potential tumor marker. Ferritin was first detected in sera of patients with severe liver disease and Hodgkin's disease by relatively insensitive immunologic techniques (3, 4). An 1112H pro· tein in fetal serum undetectable in adult serum was first demonstrated by Burtin et al. (5) and identified subse· quently as an isoferritin (6). The a"H protein was de· tected in the sera of 80% of children and 50% of adults with a variety of malignant disorders and in 9-18% of individuals with nonmalignant diseases (7, 8). More reo cently, ferritin was measured in normal sera by more sensitive immunoradiometric techniques (9-12), and data on elevated ferritin levels in patients with acute leukemia and Hodgkin's disease were reported (13, 14). The protein moiety of ferritin has a molecular weight of approximately 450,000 daltons (2) and was thought to consist of 24 identical subunits (1, 2); but recent studies demonstrated that the subunits were heterogeneous (15-21). Ferritin from human liver and horse spleen can be resolved into 6-7 isoferritin components by isoelectric focusing (16-19), and this heterogeneity is apparently related to the subunit composition of the assembled ferritin molecules (20, 21). Components of ferritin with unusually low isoelectric points were first demonstrated in cultured cells from human and animal tumors (22-24) and more recently in human fetal liver, hepatomas, and placenta (16, 25). These acidic isoferritins 5 were termed "carcinofetal" (16), but the more recent demonstration of similar acidic components in normal adult heart, pancreas, and kidney (19) suggested that this designation should not be used unqualifiedly. No immunologic dif-

ferences were demonstrated between normal isoferritins or between normal and acidic isoferritins. The marked increase in ferritin content of several mammary carcinomas (18) suggested that measurement of serum ferritin levels may be useful in the clinical evaluation and management of patients with these tumors. In this communication, we present our radioimmunoassay (RIA) technique for measuring ferritin and our results in patients with breast cancer and some inflammatory states. We also present evidence for an immunologic difference between normal and acidic isoferritins. MATERIALS AND METHODS

Ferritins.-Ferritin was isolated from hepatic metastases of a breast tumor and from a normal human heart by the immunoadsorbent method described in (18). The isoferritin composition of the preparation used as the radioactive standard (DEAE 85 I) and a fraction enriched in acidic isoferritins (DEAE 85 III) were described (18). Isoelectric focusing of heart ferritin in polyacrylamide gel discs (18) revealed all acidic isoferritins and none of the major isoferritin components of normal adult liver. All heart isoferritin bands gave a positive Prussian blue stain for iron (18) and a positive immunoprecipitate with antiferritin antibodies (19). A detailed account of the chemical and immunologic properties of heart ferritin will be presented elsewhere. Normal liver ferritin was a gift from Drs. Y. Niitsu and I. Listowsky (26). Antisera.-The rabbit antiserum to ferritin, used as the first antibody in the RIA, was prepared against DEAE 85 I as described (18). The precipitin bands produced by the reaction of this antibody with several ferritin preparations, DEAE 85 I, DEAE 85 III, normal liver ferritin, and heart ferritin fused completely. The second antibody was obtained by immunization of sheep with rabbit IgG prepared by chromatography on DEAE cellulose (27). Rabbit IgG (400 p.g) in I ml saline was mixed with an equal volume of complete Freund's adjuvant containing 2 mg M tuberculosis H 37 RA (Difco Laboratories, Inc., Detroit, Mich.). The sheep was given 0.5 ml of this mixture im at two sites; the other I ml was inReceived May 1, 1975; accepted June 17, 1975. Supported by Public Health Service contract NOI CB23871 from the Division of Cancer Biology and Diagnosis of the National Can· cer Institute (NCI) , and by grant I-POI CAI33303 from the NCI. 3 Departments of Medicine, Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, N.Y. 10461. 4 We thank Dr. Mitchell Gail, Biometry Branch, National Cancer Institute, Bethesda, Md., and Dr. Sylvia Wassertheil, Department of Community Health, Albert Einstein College of Medicine, Bronx, N.Y., for their advice and assistance with the statistical analysis of the data. We also thank Mrs. Angela Doyle for her excellent technical help, Mrs. Vivian Wartti for aid in collection of sera and tumor specimens, and Mr. Nicholas Gutfeld of the Computer Center of the Albert Einstein College of Medicine for his adaptation of a computer program for the radioimmunoassay and calculation of the SD. 5 Acidic isoferritins have a more rapid electrophoretic mobility (lower isoelectric point) than the most anodal component of human liver ferritin. 1

2

JOURNAL OF THE NATIONAL CANCER INSTITUTE, VOL. 55, NO.4, OCTOBER 1975

791

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SUMMARY-Ferritins are iron·containing proteins found in normal tissues; they increase in concentration in many tumors and the blood of tumor·bearing individuals. We utilized a double·antibody radioimmunoassay for measurement of serum ferritin and defined the upper limit of normal as 146 ng/ml for women (mean 34 ng/ml) and 193 ng/ml for men (mean 93 ng/ml). Serum ferritin levels exceeded these limits in pre· operative sera of 41 % of women with mammary carcinoma (mean 199 ng/ml) and in 67% of women with locally recur· rent or metastatic mammary carcinoma (mean 671 ng/ml). Individuals with hepatic inflammatory states are known to have high serum ferritin, and ferritin was increased in 43'% of patients with hepatitis or cirrhosis (mean 364 ng/ml) and in 13% of patients with ulcerative colitis or gastroduodenal ulcers (mean 106 ng/ml). Measurement of serum ferritin may be useful in evaluation of patients with breast cancer and in monitoring their response to therapy.-J Natl Cancer Inst 55:

792

MARCUS AND ZINBERG TABLE

I.-RIA and calculation altke

x value (ng ferritin)

1.0 2.5 5.0 7.5 10.0 15.0 20.0

SD

SE

(% difference) ±9.0 ±4.0

±5.0 ±7.9

±7.0 ±3.3 ±0.6

computer system to ascertain the following: 1) the coefficients of the line y = ax + b; 2) the index of determination (the square of the correlation coefficient); 3) the predicted y value; and 4) the percentage difference between the actual and predicted y values. The y values represented net counts per minute (cpm) in the precipitate, and the x values represented nanograms of unlabeled ferritin. All the curves conformed to a straight line and had correlation coefficients averaging 0.92. To determine how closely the predicted cpm agreed with the observed experimental values, we analyzed six standard curves to find the SD of the percentage difference at each y value. The results of these calculations are presented in table 1. RESULTS Normal Individuals

Serum ferritin levels in 57 normal men and women are in text-figure I and table 2. The lower limit of sensitivity of the assay was I ng and sera containing less than I ng/O.l ml sample were arbitrarily assigned a value of 10 ng/m!. Because of this uncertainty about the true distribution of values below 10 ng/ml and because the histograms in text-figure I do not appear to have a gaussian distribution, nonparametric methods were used to set an upper limit of normal (29). A value of 193 ng/ml represented an upper 94.6% tolerance limit on 95% of the population for males, and 146 ng/ml was an upper 97.5% tolerance limit on 95% of the population for females. Breast Cancer

Serum ferritin values exceeded the upper limits of normal in 41% (14/38) of women who subsequently underwent mastectomy for a mammary carcinoma and in 67% (65/97) of women with locally recurrent or metastatic

~+I

NORMAL FEMALE

10

~

5

~:;

0

a ~

015

NORMAL MALE

~

'"~ 10 z

o I.-Distribution of serum ferritin values in normal men and women.

TEXT-FIGURE

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jected at multiple intracutaneous sites; 0.5 ml pertussis vaccine (Eli Lilly & Co., Indianapolis, Ind.) was administered sc at a separate site. Labeling of ferritin.-Ferritin was labeled by the method of Hunter (28). The initial reaction mixture contained 0.02 ml 0.5 M sodium phosphate buffer (pH 7.5), 12-25p.g ferritin (DEAE I) in 0.05 ml potassium phosphate buffer (0.01 M, pH 7.8), and 1 mCi '25 I in 0.011 ml (iodide; carrier free; sp act 100 mCi/ml; Amersham-Searle Corp., DesPlaines, Ill.). After the addition of 0.01 ml chloramine-T (2 mg/ml) (Eastman Kodak Co., Rochester, N.Y.), the reaction proceeded for I minute at room temperature, and then 0.05 ml sodium metabisulfite (2 mg/ml) and 0.005 ml 1 M potassium iodide were added. The labeled ferritin was separated from free 1251 by passage through a 1 X 20-cm column of Sephadex G-25 (Pharmacia Fine Chemicals, Inc., Piscataway, N.J.) which was prepared by being washed with 20 ml TEN buffer [0.02 M Tris (pH 7.6), 0.001 M EDT A, 01 M NaCl], 0.5 ml TEN buffer containing 10 mg bovine serum albumin (BSA) /ml, and 20 ml TEN buffer containing I mg BSA/ml. After we removed 0.002 ml from the reaction mixture to calculate recovery of ferritin from the column, the remainder of the sample was applied to the column developed with TEN buffer containing I mg BSA/ml. Fractions of 0.5 ml were collected and the radioactive peaks were detected by a Geiger-Miiller counter. The protein peak usually emerged in fractions 8-10, and the free 1251 started to emerge in fractions 17-20. We determined the percentage of radioactivity precipitable by trichloroacetic acid (TCA) by mixing aliquots of diluted column fractions with 3 ml cold TCA, allowing precipitation to take place for 30 minutes at 4° C, passing the mixture through a type-IA Millipore filter (Millipore Corp., Bedford, Mass.), and washing the filter with cold TCA. The radioactivity of the filters was measured in an automatic gamma counter (NuclearChicago Corp., DesPlaines, Ill.). In general, 60-80% of the radioactivity in the protein peaks was precipitable by TCA, and the recovery of TCA-precipitable counts from the column ranged between 70 and 100%. Approximately 75-100% of these counts were precipitated by antibodies in the RIA. Radioimmunoassay.-All dilutions for the RIA were made with phosphate-buffered saline (PBS)-BSA [0.02 M phosphate (pH 7.3) 0.9% NaCI] buffer containing 10 mg BSA/ml. The sample to be assayed (0.05-0.1 ml) was mixed with 1-2 ng of labeled ferritin. Diluted rabbit antiserum to ferritin (0.1 ml) (1 :50,000-1 :75,000) was added, and the total volume was brought to 1 ml with PBS-BSA buffer. After incubation at 4° C for approximately 24 hours, 0.025 mil: 10 dilution of normal rabbit serum was added as a carrier for the precipitation, the tubes were mixed, 0.05-0.1 ml sheep antiserum to rabbit IgG was added, and the tubes were incubated overnight at 4° C. On the final day of the assay, 1 ml cold PBS-BSA buffer was added to each tube, and, after being mixed, the tubes were centrifuged for 45 minutes at 1,300xg at 4° C, the supernatants were decanted, and the inverted tubes were drained on absorbent paper and placed in a gamma counter for measurement of radioactivity. All samples were run in duplicate and known quantities of unlabeled ferritin, 1-20 ng, were included in each assay as a reference. We generated standard curves by fitting a straight line to the standard data points in the least-squares sense. An on-line regression program was used on a Xerox Sigma 6

793

MEASUREMENT OF SERUM FERRITIN BY RADIOIMMUNOASSAY

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2000 1000

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200 100

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2.-Serum ferritin values in normal individuals, women with breast cancer, and patients with nonmalignant hepatic or gastrointestinal (GI) disorders. In nonmalignant GI category, 0 represents patients with hepatitis or cirrhosis and. represents patients with ulcerative colitis or gastroduodenal disorders. Dashed lines indicate upper limits of normal-146 ng/ml for women and 193 ng/ml for men.

TEXT-FIGURE

mammary carcinoma (text-fig. 2). The distribution of "normal" values in women with breast cancer differs significantly from normal women; i.e., ferritin levels below 10 ng/ml were found in 45% of normal women, 25% of preoperative patients, and 2% with recurrent or metastatic disease (text-fig. 2).

control group had elevated ferritin values (text-fig. 2). Most abnormal values were in patients with hepatic diseases, 43% (18/42), in contrast to 13% (4/31) of the patients with ulcerative colitis or gastroduodenal disorders. Hematologic Disorders

A few patients with leukemia and lymphoproliferative diseases were examined; elevated ferritin levels were

Inflammatory Diseases

In view of previous reports of elevated fern tin levels in patients with liver disease, we studied a control group with nonmalignant hepatic and GI disorders. This group included II patients with alcoholic cirrhosis, 31 with chronic hepatitis, 14 with gastroduodenal ulcer, and 19 with ulcerative colitis. Approximately 30% (22/73) of this 2.-Serum ferritin values in normal individuals, women with breast cancer, and patients with nonmalignant hepatic and GI disorders

TABLE

10,000

~ 8,000 ii:

~

"-

z

~

"-

Subjects Normal women__ ________ Normal men____________ Women with breast cancer Preoperative_ _ _ _ _____ _ Recurrent_____________ Patients with nonmalignant diseases Hepatic_______________

(}I___________________

Number of patients

Percent elevated

Ferritin (ng/ml) Mean

117

34 93

57

Range

6,000

HEART 85m

4,000

U

I-

Z 2,000

851 NORMAL LIVER

10-146 10-193

38 97

41 67

199 671

10-1,394 10-5,875

42 31

43 13

364 106

10-3,232 10-786

FERRITIN (ng/ml) 3.-Comparison of the inhibitory activities of normal and acidic isoferritins in ferritin RIA. Normal ferritins are DEAE 85 I and normal liver ferritin. Acidic ferritins are DEAE 85 III and heart ferritin. Points were fitted to straight line by computer program.

TEXT-FIGURE

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~

00

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300

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..

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I

600

794

MARCUS AND ZINBERG

found in 6/13 patients with Hodgkin's disease, 6/9 with leukemia, and 6/9 with multiple myeloma. Many of these patients had extremely high ferritin levels. The abnormal values for the myeloma patients were 223; 619; 891; 4,604; 10,592; 15,256. All 4 patients with acute myeloid leukemia had high ferritin levels: 1,153; 9,964; 12,464; 14,856 ng/ml. Acidic Ferritins

DISCUSSION

Immunoradiometric assays for ferritin used by several groups (9-12) differ from our procedure in that these investigators used insolubilized antigens and radiolabeled antibodies rather than labeled antigens. Despite these differences, there is reasonable agreement on the upper limits of ferritin in normal sera: 180-186 ng/ml for men and 143-162 ng/ml for women (12,14). Fuccaro-Decaens et al. (30), using a double-antibody RIA similar to ours, reported an upper limit of 92 ng/ml for women and 297 ng / ml for men. Serum ferritin levels may be influenced by a number of factors, including total-body iron stores, release of ferritin from inflamed tissues, and production of ferritin by proliferating tumor cells. A direct correlation between serum ferritin levels and the total-body iron stores was demonstrated (9, 10,31), and the impairment of erythropoiesis associated with chronic inflammation, infection, or malignant states (32, 33) would be expected to increase iron stores and ferritin levels. Elevated ferritin levels associated with liver disease (3, 4, 34) are probably caused by leakage of ferritin from inflamed hepatic tissue. The only direct evidence for increased synthesis of ferritin by a mali?;nant cell is the study by White et al. (35) on acute myeloid leukemia. Tissue culture lines derived from tumors, HeLa and KB, synthesize ferritin (22, 23, 36), and ferritin has been detected in mammary carcinomas by immunofluorescence (18). Despite these uncertainties about the origin(s) of serum ferritin, measurement of this protein may have empirical value in cancer immunodiagnosis (8, 13, 14,37,38). The low incidence of elevated ferritin levels in patients with small, localized mammary carcinomas and the high ferritin values in patients with liver disease preclude the use of this assay for screening patients or in the differential diagnosis of a suspicious breast lesion. The assay may be useful in evaluating the prognosis of patients with documented breast cancer, in detecting the persistence of tumor after mastectomy, in detecting recurrences of tumor before they are clinically evident, and in monitoring therapy. We are now carrying out longitudinal studies of patients to evaluate these potential clinical applications. Elevated serum ferritin levels were found in virtually all untreated patients with acute myeloid leukemia (13), and the recent report of normal ferritin levels in patients after successful chemotherapy (39) suggests that ferritin may be usefwl in monitoring therapy of patients with acute leukemia. The finding of high ferritin levels in patients with clinically apparent, severe liver disease agrees with previous reports (3, 4, 34) and

REFERENCES

(1) CRICHTON RR: Ferritin: Structure synthesis and function. N Engl J Med 284: 1413-1422, 1971 (2) - - - : Structure and function of ferritin. Angew Chern [Engl] 12:57-65, 1973 (3) REISSMANN KR, DIETRICH MR: On the presence of ferritin in the peripheral blood of patients with hepatocellular disease. J Clin Invest 35:588--595, 1956 (4) AUNGST CW: Ferritin in body fluids. J Lab Clin Med 71:517522, 1968 (5) BURTIN P, VON KLEIST S, BUFFE D: I. Etude immunoelectrophoretique des antigcnes du serum foetal humain absents du serum adulte. Bull Soc Chim Bioi 49:1389--1398, 1967 (6) BUFFE D, RIMBAUT C, FUCCARO C, et al: Isolement et caracterisation d'une 2 ferroproteine: La globuline ll'2H. Ann Inst Pasteur (Paris) 123:29--42, 1972 (7) BUFFE D, RIMBAUT C, LEMERLE J, et al: Presence d'une ferroproteine d'origine tissulaire l'alpha-2H dans Ie serum des enfants porteurs de tumeurs. Int J Cancer 5:85-87, 1970 (8) RIMBAUT C: "L'mH globuline" glyco-proteine reactionelle serique d'origine hepatique: Ses rapports avec les affections malignes. Bull Cancer (Paris) 60:411-420, 1973 (9) ADDISON GM, BEAMISH MR, HALES CN, et al: An immunoradiometric assay for ferritin in the serum of normal subjects and patients with iron deficiency and iron overload. J Clin Pathol 25:326-329, 1972 (10) JACOBS A, MILLER F, WORWOOD M, et al: Ferritin in the serum of normal subjects and patients with iron deficiency and iron overload. Br Med J 4:206-208, 1972

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Although no immunologic differences between normal and acidic ferritins were revealed by double diffusion in agarose gel, they were readily demonstrated by the RIA (text-fig. 3). Acidic ferritin from normal heart or a mammary carcinoma (DEAE85 III) was much less effective than DEAE 85 I or normal liver ferritin in competing with DEAE 85 I for the antibody.

should not present a difficult clinical problem in evaluation of women with documented breast cancer. Carcinoembryonic antigen (CEA) is the only tumor marker other than ferritin that is positive in an appreciable percentage of patients with common epithelial tumors, and CEA levels are also elevated in many nonmalignant states (40). The primary use of CEA at present is also in the evaluation and follow-up of patients with documented malignancies (40). The difference in slope between inhibition curves of acidic and normal ferritins indicates that these subclasses of ferritin do not possess identical antigenic determinants. The determinants are highly cross-reactive and the differences are not demonstrated by double diffusion in agar gel-a technique requiring relatively concentrated solutions of antigen and antibody. The differences in affinity (association constant) of the two antigens for the antibody are revealed by the use of limited quantities of reagents in the RIA. Drysdale (personal communication) also found that immunologic dissimilarities between normal and acidic ferritins can be demonstrated by RIA. The chemical basis of the charge differences between normal and acidic ferritins is not known, but differences between their subunits were recently demonstrated (41). The wide range of serum ferritin values in normal women decreases the sensitivity of the RIA for tumor immunodiagnosis, because a woman with a baseline normal ferritin value of 10-15 ng/ml could have a tenfold increase and her serum ferritin would still fall within the normal range. In addition, the relatively weak inhibitory activity of acidic ferritin (text-fig. 3) indicates that our present RIA would underestimate the ferritin level of a serum containing appreciable quantities of acidic isoferritins. Nothing is known at present about the isoferritin profile of normal or pathologic sera. If the isoferritin of normal serum resembles that of human liver or spleen (organs rich in ferritin) and if tumors other than hepatomas synthesize appreciable quantities of acidic ferritin, a considerable increase in specificity and sensitivity would be provided by an RIA specific for acidic ferritin. To answer these questions, we are currently studying the isoferritin profile of serum.

MEASUREMENT OF SERUM FERRITIN BY RADIOIMMUNOASSAY

(29)

(JO)

(Jl)

(J2)

(JJ) (34)

(35) (J6)

(37) (38)

(39) (40)

(41)

mental Immunology, 2d cd. (Weir DM, ed.). Oxford, Blackwell Scientific Pub, 1973, pp 17.1-17.36 SIEGEL S: Nonparametric Statistics for the Behavioral Sciences. New York, McGraw-Hill, 1956, pp 202-210 FUCCARO-DECAENS C, MERIADEC B, BUFFE D, et al: Etude comparative de deux techniques sensibles de dosage immunologique: Le dosage radioimmunologique et Ie dosage enzymoimmunologique appliques it la detection de l'a2Hglobuline serique. Ann Immunol 125C:747-761, 1974 LIPSCHITZ DA, COOK JD, FINCH CA: A clinical evaluation of serum ferritin as an index of iron stores. N Engl J Med 290:1213-1216, 1974 CARTWRIGHT GE: The anemia of chronic disorders. Semin Hematol 3:351-375, 1966 ZUCKER S, FRIEDMAN S, LYSIK RM: Bone marrow erythropoiesis in the anemia of infection inflammation malignancy. ] Clin Invest 53: II 32-II38, 1974 MARTIN ]P, CHARLIONET R, ROPARTZ C: The presence of alpha-2H in sera from patients with malignant haemopathies and cirrhosis. Rev Eur Etud Clin BioI 16:266--268, 1971 WHITE GP, WORWOOD M, PARRY DH, et al: Ferritin synthesis in normal and leukaemic leukocytes. Nature 250:584-586, 1974 CHU LL, FINEBERG RA: On the mechanism of iron-induced synthesis of apoferritin in HeLa cells. J BioI Chern 244: 3847-3854, 1969 BIEBER CP, BIEBER MM: Detection of ferritin as a circulating tumor-associated antigen in Hodgkin's disease. Nat! Cancer Inst Monogr 36:147-157, 1973 ESHHAR Z, ORDER SE, KATZ DH: Ferritin, a Hodgkin's diseaseassociated antigen. Proc Natl Acad Sci USA 71:3956--3960, 1974 PARRY DH, WORWOOD M, JACOBS A: Serum ferritin in acute leukaemia at presentation and during remission. Br Med J 1:245-247, 1975 NEVILLE AM, LAURENCE DJ: Report of the workshop on the carcinoembryonic antigen (CEA): The present position and proposals for future investigation. Int J Cancer 14:1-18, 1974 ISHITANI K, NUTSU Y, LISTOWSKY I: Ferritin subunit structure and heterogeneity. In Proteins of Iron Storage and Transport (Crichton R, ed.). Amsterdam, North Holland Publ Co., 1975. In press

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(11) MILES LE, LIPSCHITZ DA, BIEBER CP, et al: Measurement of serum ferritin by a 2-site immunoradiometric assay. Anal Biochem 61:209---224, 1974 (12) HALLIDAY JW, GERA KL, POWELL LW: Solid phase radioimmunoassay for serum ferritin. Clin Chim Acta 58:207-214, 1975 (13) JONES PA, MILLER I'M, WORWOOD M, et al: Ferritinaemia in leukaemia and Hodgkin's disease_ Br J Cancer 27:212-217, 1973 (14) WORWOOD M, SUMMERS M, MILLER I', et al: Ferritin in blood cells from normal subjects and patients with leukaemia. Br J Haematol 28:27-35, 1974 (15) NUTSU Y, LI~TOWSKY I: Mechanisms for the formation of ferritin oligomers. Biochemistry 12:4690--4695, 1973 (16) ALPERT E, COSTON RL, DRYSDALE ]W: Carcino-foetal human liver ferritins. Nature 242:194-195, 1973 (17) DRYSDALE ]W: Heterogeneity in tissue ferritins displayed by gel electrofocusing. Biochem J 141:627-632, 1974 (18) MARCUS DM, ZINBERG N: Isolation of ferritin from human mammary and' pancreatic carcinomas by means of antibody immunoadsorbents. Arch Biochem Biophys 162:493-501, 1974 (19) POWELL LW, ALPERT E, DRYSDALE JW, et al: Human isoferritins: Organ specific iron and apoferritin distribution. Br J Haematol 30:47-55, 1975 (20) NIITSU Y, ISHITANI K, LISTOWSKY I: Subunit heterogeneity in ferritin. Biochem Biophys Res Commun 55:II34-II40, 1973 (21) ISHITANI K, NUTSU Y, LISTOWSKY I: Characterization of the different polypeptide components and analysis of subunit assembly in ferritin. J BioI Chern 250:3142-3148, 1975 (22) RICHTER GW: Electrophoretic and serological properties of the ferritins produced by HeLa and KB cells in cultures. Br J Exp Pathol 45:88-94, 1964 (2J) - - - : Comparison of ferritins from neoplastic and nonneoplastic human cells. Nature 207:616-618, 1965 (24) RICHTER GW, LEE JC: A study of two types of ferritin from rat hepatomas. Cancer Res 30:880--888, 1970 (25) DRYSDALE JW, SINGER RM: Carcinofetal human isoferritins in placenta and HeLa cells. Cancer Res 34:3352-3354, 1974 (26) NUTSU Y, LISTOWSKY I: The distribution of iron in ferritin. Arch Biochem Biophys 158:276--281, 1973 (27) LEVY HB, SOBER HA: A simple chromatographic method for preparation of gamma globulin. Proc Soc Exp Bioi Med 103:250--252, 1960 (28) HUNTER WM: 17-Radioimmunoassay. In Handbook of Experi-

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