THE INCORPORATIONOF COPPERINTO CERULOPLASMININ VIVO: STUDIES WITH COPPER64AND COPPER67*

By IRMIN STERNLIEB, ANATOLG. MORELL, WALTERD. TUCKER,MARGARETW. GREENEAND I. HERBERTSCHEINBERG

(From the Department of Medicine, Albert Einstein College of Medicine, and Bronx Munici-pal Hospital Center, New York, N. Y., and Radioisotope Development Section,

Hot Laboratory Division, Brookhaven National Laboratory, Upton, N. Y.)

(Submitted for publication April 28, 1961; accepted May 29, 1961)

The fact that absence or deficiency of the plasmacopper-protein, ceruloplasmin, is almost alwaysassociated with the excessive deposits of tissuecopper which characterize Wilson's disease hassuggested that ceruloplasmin regulates copperabsorption or excretion (1). Based on the invitro demonstration of reversible dissociation ofcopper from ceruloplasmin, a hypothesis wasproposed of how such regulation could be effected(2). This stated that if reversible dissociation ofcopper from ceruloplasmin occurred in the intes-tinal wall in vivo, a concentration of copper ionswould result which could oppose the net transferof copper ions from the intestinal lumen into theblood. The three experiments reported in thispaper were designed to determine whether ex-change of ceruloplasmin copper for ionic copper,which is equivalent to reversible dissociation ofceruloplasmin copper, takes place in vivo.

In the first experiment, exchange of cerulo-plasmin copper was looked for in patients withWilson's disease in whom virtually no synthesisof the protein was occurring. The interpretationof this experiment required knowledge of whetherthe catabolism of ceruloplasmin was detectablydifferent in a normal individual from its catabo-lism in patients with Wilson's disease. A com-parison of the half-life of ceruloplasmin, as onemeasure of its catabolism, was made in these twotypes of individuals in the second experiment. Inthe third experiment, the incorporation of ioniccopper into ceruloplasmin was studied in an indi-vidual with normal copper metabolism in such a

* Presented in part at the Eastern Section Meeting ofthe American Federation for Clinical Research, January6, 1961, Bethesda, Md. This work was supported inpart by grants from the National Institute of Arthritisand Metabolic Diseases of the United States PublicHealth Service (A-1059), and the Life Insurance Medi-cal Research Fund (G-60-54).

way that incorporation occurring during synthe-sis of ceruloplasmin could be differentiated fromthat resulting from copper exchange.

All of our results were compatible with theconclusions that copper entered the ceruloplasminmolecule only at the time of synthesis, and thatexchange of ceruloplasmin copper with ioniccopper did not occur in vivo. Since it was acorollary of these conclusions that a given copperatom was a label which remained on a cerulo-plasmin molecule throughout its life span, wewere able to calculate the rate of turnover ofceruloplasmin, and its copper, in an individualwith normal copper metabolism.

EXPERIMENTAL

Materials and methods

Ceruloplasmin. Two solutions (CH-157 and CH-240),which contained 2.9 and 2.1 g of purified human ceru-loplasmin per 100 ml, respectively, were obtained fromthe Ortho Research Foundation, Raritan, N. J.1 Bothpreparations were sterile, nontoxic, nonpyrogenic andtheir intravenous administration to human beings pro-duced no ill effects.

Copper'. Sterile solutions of cupric" acetate were ob-tained from Abbott Laboratories, Oak Ridge, Tenn. Thehalf-life of this isotope is 12.8 hours (3).

Copper". Copperr was produced in the 60-inch cyclo-tron of the Brookhaven National Laboratory by the fol-lowing (a, p) reaction: Ni' + He4-- Cu67 + H. Sincethere is only 1 per cent of Ni' in naturally occurringnickel, material enriched to 96 per cent Ni' was obtainedfrom the Isotope Division of the Oak Ridge National

1 Through the cooperation of Sam T. Gibson, Director,American National Red Cross Blood Program, andJames A. McComb, John M. Newell and Lewis H. Lar-sen of the Biologic Laboratories, Department of PublicHealth of the Commonwealth of Massachusetts, we ob-tained Cohn fraction IV-1 from human plasma. Cerulo-plasmin was prepared from fraction IV-1 under the di-rection of H. 0. Singher of the Ortho Research Founda-tion, Raritan, N. J.

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INCORPORATIONOF COPPERINTO CERULOPLASMININ VIVO

TABLE I

Labeling of ceruloplasmin with copper67 by M3ethod A

Ceruloplasmin 0.85% Ascorbic Cu67-Expt. Subj. (CH-240) NaCi acid nitrate* Cu67 Final pH

ml g ml mg ml gI G.G. 63.0 1.32 240 785 2.3 71 6.18I D.S. 63.0 1.32 340 785 2.0 62 6.45

* This solution contained 31 ,ug copper/ml with a specific activity of 1.21 ycc/pug.Laboratory and used as the target. Although enrichednickel' was relatively expensive, it could be recoveredwith a yield of about 98 per cent and reused.

To prepare approximately 1 to 2 mc of copper", 60 mgof the nickel' was electroplated as metal onto gold foilthat had been silver-soldered to a copper target holder.(The area covered by the nickel was roughly / X %inch. It varied in thickness from 0.007 to 0.010inch, which was sufficient to absorb the entire alphabeam.) The target assembly was mounted on a water-cooled probe that was inserted into the vacuum chamberof the cyclotron, and the nickel' target was bombardedby the internal beam of 40 Mev alpha particles. At thecurrent of 50 to 60 /ua which resulted, bombardments of10 to 12 hours were required. For example, 1.4 mc wasproduced in a 500 pa-hour bombardment, which corre-sponded to a thick-target yield of 2.8 ,uc per ga-hour.

The target was removed from the cyclotron and thegold foil was separated from the target holder by melt-ing the silver solder. The bombarded nickel was dis-solved in a few drops of nitric acid. After addition of0.5 mg of copper carrier, hold-back carriers of indium,cadmium and cobalt, and 2 or 3 drops of concentratedsulfuric acid, the copper was electroplated at 80° C for30 minutes onto a platinum electrode. The copper wasdissolved from the platinum with nitric acid and wasused without sterilization. The processing yield wasestimated to be at least 95 per cent. The only radio-contaminant present in significant amount was copper',which constituted initially about 7 per cent of the totalradioactivity.

Nitric acid was removed from the solution which re-mained after the electroplating of copper by evaporationto fumes of sulfur trioxide. The residue was dilutedwith water, made alkaline with concentrated ammonia,and the nickel' was recovered by electroplating.

The half-life of copper' was determined to be 61.8 ± 0.8hours. To our knowledge the experiments reportedin this paper represent the first use of this isotope inbiological research.

Radioactive labeling of ceruloplasmin copper

1. Method A. To a solution of purified ceruloplasminin physiological saline, ascorbic acid and then copper'nitrate were added. After standing at room temperaturefor 1.5 hours, approximately 35 per cent of the copper'was found to have been incorporated into the cerulo-plasmin of each solution by exchange. The amounts of

material used for the two preparations are given inTable I.

2. Method B. Two male volunteers, aged 50 and 64,received 1.0 mg of ethinyl estradiol (Estinyl) daily for5 days in order to elevate their plasma concentrationsof ceruloplasmin (4, 5). By intravenous injection theywere then given, respectively, 3.45 and 4.77 mg of Cu¶,with a specific activity of 105 /Ac per mg, as cupric" ni-trate.2 Forty hours later a phlebotomy was performedon each volunteer yielding, in a total of 450 ml of plasma,286 mg of labeled ceruloplasmin. This contained 88 l.egof ceruloplasmin-bound Cu8, and only 1.3 ,ug of nonceru-loplasmin Cu. Thus, this material was of much greatervalue in studying ceruloplasmin metabolism in a controlsubject than ceruloplasmin labeled by Method A whichalways contained a large amount of nonceruloplasmincopper'. Although the incidental administration of thelatter was unimportant in the experiment in the 2 pa-tients with Wilson's disease, who cannot synthesizeceruloplasmin, nonceruloplasmin copper would have en-tered newly synthesized ceruloplasmin in control sub-jects in vivo and made Experiment II impossible.

Quantitative determination of ceruloplasmin. Cerulo-plasmin concentrations in serum were measured by de-termining the oxidase activity of serum toward para-phenylenediamine (2).

Quantitative determination of radioactive copper" andcopper. Four-ml aliquots of serum, or smaller ali-quots brought to 4 ml by addition of normal saline, werecounted in a Nuclear-Chicago DS5-5 well-type scintil-lation counter by means of a 161A scaler. Discrimina-tion between the 0.184 Mev gammarays of Cu' (3) andthe 0.51 Mev annihilation gamma rays (6) of Cu" waseffected by inclusion of a Nuclear-Chicago 1810 radia-tion analyzer in the circuit. Correction for backgroundand physical decay yielded net corrected counts per min-ute per milliliter of serum due to each isotope.

Separation of nonceruloplasmin copper. In order todetermine the proportion of the radioisotopic copper inserum bound to ceruloplasmin in Experiments I and III,the nonceruloplasmin copper in the serum was made toreact with sodium diethyldithiocarbamate (DTC) and

2 In order to keep the copper in solution, each dose ofCue was dissolved in a mixture of 60 ml Amigen (5 percent enzymic casein hydrolysate and 5 per cent dextrose,in water) and 0.5 ml of 7.5 per cent sodium bicarbonate.The solutions were clear, blue, and had a pH of 5.2.Neither produced any untoward reaction when infused.

1835

STERNLIEB, MORELL, TUCKER, GREENEAND SCHEINBERG

mm./imin&mL0.4000

3000 _

0.2000

0.10001_

0.5001-

0.2501_

W.OOF

o.5oL

CPM/mL

AOD./minAnl.0.200OF

0.0ooor

0.500o

0.2501

0.100

HOURS

O-Cud°-Oxidase Activity

HOURS

FIG. 1. HALF-LIFE OF COPPER"T-LABELED CERULOPLASMINOBTAINED FROMTHE RATES OF DISAPPEARANCEOF

OXIDASE ACTIVITY ANDRADIOACTIVITY OF SERUMIN TWOACERULOPLASMINEMICPATIENTS WITH WILSON'S DIS-

EASE, G.G. (LEFT) AND D.S. (RIGhT).

the colloidal Cu-DTC complex was adsorbed on a col-umn of activated charcoal, as follows. After dilutionof 3 ml of serum with an equal volume of normal saline,0.6 ml of a 0.1 per cent solution of DTC in saline was

added. The mixture was passed through a column of100 mg of activated charcoal (Norit-A). The 4 ml ofeffluent which followed the 1.5 ml fore-run had the same

concentration of ceruloplasmin as that contained in the6.6 ml of solution passed over the column, and containedneither free copper nor free DTC.

RESULTS

Experiment I. We infused 1.32 g of cerulo-plasmin, labeled by Method A with copper6, intoeach of two patients with Wilson's disease withoutserum ceruloplasmin. There were no untowardeffects on pulse, respiration, blood pressure, or

venous clotting and prothrombin times. Serialmeasurements of the serum concentration of the

protein were obtained both by 1) determining theoxidase activity of the serum toward paraphenyl-enediamine and 2) counting the radioactivityof the serum. The latter was approximatelyequivalent to determining coppery in cerulo-plasmin since, beyond 24 hours after infusion, lessthan 10 per cent of copper67 in serum was non-

ceruloplasmin copper67. The results are given inFigure 1. The values for the half-life of cerulo-plasmin given in the figure and in Table II were

calculated independently from the results of boththe enzymatic and radioactive measurements bythe method of least squares. The volumes of dis-tribution of the infused protein (Table II) were

obtained by dividing the amounts administered bythe serum concentration of ceruloplasmin at zero

time determined by extrapolation.

LE II

Volumes of distribution, half-lives and daily turnover rate of ceruloplasmin and ceruloplasmin copper

Dailyturnoverrate of

Volume Oxidase Radio- cerulo-of distri- activity activity Daily turnover rate of plasmin

Expt. Subj. bution half-life half-life ceruloplasmin* copper

L days days S mg mgI G.G. (Wilson's 3.77 4.16 4.39 15.7 207.2t 0.704

disease)

I D.S. (Wilson's 3.41 4.80 5.15 13.4 176.9t 0.601disease)

II J.M. (control) 3.97 5.40 12.9 141.9 0.483

* Calculated from radioactivity measurements.t Immediately after infusion.

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INCORPORATIONOF COPPERINTO CERULOPLASMININ VIVO

Experiment II. Into a normal male volunteer,aged 22, 450 ml of plasma containing 286 mg ofcopper 67-labeled ceruloplasmin (Method B) was

infused. The concentration of the copper67-labeledceruloplasmin was measured daily by determiningthe radioactivity of whole serum, and these re-

sults were used to calculate the half-life of cerulo-plasmin and its volume of distribution (Figure 2and Table II). In contrast to the previous ex-

periment, we could not also calculate these valuesfrom measurements of oxidase activity, since theoxidase activity of the infused, labeled ceruloplas-min could not be distinguished from that of thesubject's own ceruloplasmin which was continu-ally being synthesized.

Experiment III. A 39 year old female withHuntington's chorea and no detectable abnormalityof copper metabolism was given 2 mg of copper64orally, as cupric64 acetate, on three different andwidely separated occasions. 1) The first dosewas administered when the patient's ceruloplasminconcentration was at a normal value, 26.7 mg per

100 ml of serum. 2) The second was given 5 daysafter she had received, for 15 days, 1 mg ethinylestradiol daily. The estrogen served to increasethe rate of synthesis of ceruloplasmin and to raise

CPM/ml.600-_6500 - * Cu 6400300

200 _ 0

Half -Life 5.40 days100 _

SOr _1_ _I- I_ !.24 48 72 96

HOURS120 144 168

FIG. 2. HALF-LIFE OF COPPER67-LABELEDCERULOPLASMIN

OBTAINED FROMTHE RATE OF DISAPPEARANCEOF THE RA-

DIOACTIVITY OF SERUMIN A CONTROLSUBJECT (J.M.).

its serum concentration from 29.2 to 51.9 mg per

100 ml (4, 5). 3) The third administration of

copper occurred 1 day after she had received an

infusion of 1.39 g of purified human ceruloplasmin(CH-157). The infusion did not, of course, in-crease the rate of synthesis of ceruloplasmin, butdid raise its serum concentration from 25.5 to74.1 mg per 100 ml. The concentration of serum

copper64 and its proportion in ceruloplasmin were

measured at intervals (Figure 3) after each doseof copper6.

10 x X T ESTROGENP,

X CERULOPLASMIN:51.9 mg/iOOmi

9 _

Fraction of CONTROL.Fractionof CERULOPLASMIN:

CU64 in 7 x 26.7mg /iOOmI

Ceruloplosmin6

from Iml x

of serum o/Tl.v. CERULOPLASMIN

x 106 4 t CERULOPLASMIN:4 ~~~~~~~~~~~~~74.1lmg/ioomi-

x

I

0 10 20 30 40 50

HOURSFIG. 3. INCORPORATIONOF COPPER' INTO CERULOPLASMININ A CONTROLSUBJECT (P.N.)

UNDERTHREE DIFFERENT EXPERIMENTALCONDITIONS.

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STERNLIEB, MORELL, TUCKER, GREENEAND SCHEINBERG

DISCUSSION

Experiment I indicated that in vivo exchangeof ceruloplasmin copper did not occur in either oftwo patients with Wilson's disease. Entire ceru-

loplasmin molecules disappeared at a rate not sig-nificantly different from that at which cerulo-plasmin-copper67 disappeared from the plasma.3The latter rate should have exceeded the formerif exchange had occured between ceruloplasmin-copper67 and the "cold" copper of the body.

It is, of course, conceivable that the cerulo-plasmin which we infused could have been al-tered during fractionation, in which case theabsence of exchange might only imply that thisceruloplasmin had lost its physiological integrity.Yet by every other property which was measuredthe ceruloplasmin seemed undenatured: its labileblue color (7), its tight copper-binding, its elec-trophoretic mobility (7), its lack of antigenicityin human beings,' and its oxidase activity (10)were virtually identical with these properties ofceruloplasmin in whole plasma or serum. Never-theless, this experiment did not provide evidencethat exchange might not have occurred in normalindividuals, since the metabolism of ceruloplasminmay differ in the two groups. Consequently, we

performed Experiment II in order to compare thehalf-life of ceruloplasmin, as measured in thesepatients with Wilson's disease, with the half-lifein a normal individual. The half-life of copper67-labeled ceruloplasmin did not differ significantlyin a simultaneous test of the values determined inthe two patients and in the control subject.5 Bythis criterion, therefore, ceruloplasmin did not

3 For each patient the differences between the log (oxi-dase activity) and log (radioactivity) at each samplingtime were plotted against time, the slope of the resultingline being designated as b. Then lb-Ol/SEb = It1 was

calculated, for the indicated degrees of freedom, andfound to be t10 = 0.785 for G.G. and t, = 1.2104 for D.S.In both cases a Iti as great, or greater, might have beenfound, by chance alone, more than 50 per cent of the time.

4 Insofar as this is indicated by the constancy of thedetermined half-life and the lack of any clinical reactionincident to repeated infusions of purified ceruloplasmin inG.G. and D.S. over the past 4 years (8, 9).

5An analysis of covariance (11) was used to test thehypothesis that these three values for the half-life didnot differ significantly. An F of 3.129, with 2 and 23degrees of freedom, was obtained which could occur bychance alone more than 5 per cent of the time.

seem to be metabolized at a different rate in thepatients than in the control subject.

Rather similar values for the half-life of ceru-loplasmin in patients with Wilson's disease andin control individuals, whether determined instudies with purified ceruloplasmin or with wholeblood, have also been found previously. Thus wehave reported the half-life of ceruloplasmin to be3.2 to 4.7 days after the infusion of whole blood(8), and 5.6 to 7.2 days after administration ofunlabeled purified human ceruloplasmin (9), inpatients with Wilson's disease. Gitlin and Hughes[quoted in (12)], using I131-labeled purified hu-man ceruloplasmin, found half-lives of 5 to 7 daysin five chronically ill subjects without Wilson'sdisease. Half-lives of fibrinogen (13, 14), they-globulins (12), and albumin (15) have also beencompared in subjects with hereditary deficiency ofthe protein and in control subjects. The resultsdo not consistently indicate either similar or dif-ferent half-lives for these proteins in the two kindsof individuals.

Finally, Experiment III confirmed that in vivoexchange did not occur in an individual withoutan apparent abnormality of copper metabolism.When P.N.'s rate of synthesis of ceruloplasminwas approximately doubled by the administrationof estrogen (Study B), about twice as muchcopper64 was incorporated into the protein as incontrol Study A. However, when an even higherserum concentration of ceruloplasmin was achievedpassively, that is, by infusion (Study C), the in-corporation of copper64 into ceruloplasmin wasessentially the same as that which occurred inStudy A, indicating that exchange was not oc-curring.

Our inability to demonstrate exchange of ceru-

6 Differences in the values of half-lives may reflect themanner in which they are calculated. Thus, our valuesin Experiments I and II are calculated from all of thepoints determined after the first 24 hours. If, however,we assume that 3 days is required for equilibration ofinfused ceruloplasmin between plasma and extravascularcompartments, the results are somewhat different. Forexample, the latter assumption yields 4.80, 5.97 and 6.36days for the three values which were given in Table IIas 4.39, 5.15 and 5.40 days, respectively. For the pur-poses of the present paper, the important point is thatall values of a half-life which are to be compared shouldbe calculated from measurements made after the same

period of equilibration.

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INCORPORATIONOF COPPERINTO CERULOPLASMININ VIVO

loplasmin copper for ionic copper in any of theseexperiments is strong evidence that reversibledissociation of copper from ceruloplasmin doesnot occur in vivo. Since the hypothesis whichtwo of us have advanced [I.H.S. and A.G.M (2)]and which was briefly outlined above, was pre-dicated on the assumption that such dissociationdoes occur, this hypothesis is not likely to be true.

If, however, a copper atom labels a cerulo-plasmin molecule throughout its lifetime we canuse our results to calculate the volume of distri-bution and turnover of ceruloplasmin. The val-ues for volume of distribution are remarkablysimilar in all three subjects (Table II). Thepercentage turnover of ceruloplasmin and of cerulo-plasmin copper was calculated in all three sub-jects from the standard equation (16) (Table II),and the fact that the copper content of the proteinis 0.34 per cent (7). The value of 142 mg forthe daily turnover of ceruloplasmin in the controlsubject, J.M., corresponds to 0.5 mg of copperincorporated into ceruloplasmin daily. This mustbe close to the amount of copper which is absorbedfrom the 2 to 5 mg of copper ingested in the diet(17). Thus it seems that much of the absorbeddietary copper in a normal individual may ulti-mately be bound to a ceruloplasmin molecule of151,000 mol wt. Such bound copper should beconfined largely to the vascular compartment, andthe volume of distribution of 3.97 L found in J.M.indicates that it is.

Despite the apparent congruence of the amountof copper absorbed and that which is used dailyfor the synthesis of ceruloplasmin, our results donot greatly clarify the physiological role of cerulo-plasmin and its relationship to Wilson's disease.They yield no information, and little additionaldata are available, about: 1) the period whichelapses between absorption of dietary copper andits quantitative incorporation into ceruloplasmin;2) the fate of copper during this time; and 3)whether, and where, ceruloplasmin copper is ex-creted. Nor do they elucidate the significance ofpatients with Wilson's disease who have normalserum concentrations of ceruloplasmin, or ofhealthy heterozygous carriers of the disease whohave abnormally low serum concentrations of thisprotein (1, 18). Furthermore, the physiologicalsignificance of the in vitro demonstrations of thereversible dissociation of ceruloplasmin copper (2,

19), of the oxidase activity of ceruloplasmin (1)and of its heterogeneity (20) is also obscure.These questions require further investigation.

SUMMARY

1. The hypothesis that ceruloplasmin exchangesits copper for ionic copper in vivo was tested, bymeans of ceruloplasmin labeled with copper64 andcopper6, in two patients with Wilson's diseaseand in two control subjects.

2. No indication of exchange was found in anyof three different experiments. It appears thatcopper is incorporated into the ceruloplasmin mole-cule in vivo only at the time of synthesis of theprotein and not through exchange.

3. A corollary of these results is that copperis a lifelong label of a ceruloplasmin molecule sothat radioactive copper may be used for the de-termination of the turnover of ceruloplasmin andceruloplasmin copper.

4. The amount of copper incorporated dailyinto ceruloplasmin in a control subject correspondsclosely to the amount of copper absorbed from thedietary intake.

ACKNOWLEDGMENTS

Weare indebted to Dr. George C. Cotzias, Dr. Quen-tin B. Deming, Mr. Daniel Horowitz and Mrs. GeorgineJ. Chulstrom for their cooperation; to Drs. D. A.Brancazio, L. Kresch, H. Lubowitz, R. M. Rothman andW. Rosenbaum, and Mrs. B. Schorr, R.N. and Miss L.King, R.N. for their assistance in carrying out the meta-bolic studies; to Mrs. Shirley de Bobes-Sternberg, ofthe Division of Biostatistics, Columbia University Facultyof Medicine, for evaluating our results statistically; andto Miss Josephine Brosseau for performing all the de-terminations of oxidase activity.

REFERENCES

1. Scheinberg, I. H., and Sternlieb, I. Copper metabo-lism. Pharmacol. Rev. 1960, 12, 355.

2. Scheinberg, I. H., and Morell, A. G. Exchange ofceruloplasmin copper with ionic Cu" with refer-ence to WVilson's disease. J. clin. Invest. 1957, 36,1193.

3. Way, K., King, R. WV., McGinnis, C. L., and vanLieshout, R. Nuclear level schemes. A =40-A = 92. Washington, D. C., National Academy ofSciences, National Research Council, 1955.

4. Russ, E. M., and Raymunt, J. Influence of estrogenson total serum copper and caeruloplasmin. Proc.Soc. exp. Biol. (N. Y.) 1956, 92, 465.

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STERNLIEB, MORELL, TUCKER, GREENEAND SCHEINBERG

5. German, J. L., III, and Bearn, A. G. Effect of es-trogens on copper metabolism in Wilson's disease.J. clin. Invest. 1961, 40, 445.

6. Quimby, E. H., Feitelberg, S., and Silver, S. inRadioactive Isotopes in Clinical Practice. Phila-delphia, Lea and Febiger, 1958, chap. 6, p. 68.

7. Holmberg, C. G., and Laurell, C.-B. Investigationsin serum copper II. Isolation of the copper con-taining protein, and a description of some of itsproperties. Acta chem. scand. 1948, 2, 550.

8. Scheinberg, I. H., Harris, R. S., Morell, A. G., andDubin, D. Some aspects of the relation of cerulo-plasmin to Wilson's disease. Neurology 1958, 8,suppl. 1, 44.

9. Scheinberg, I. H., and Sternlieb, I. Environmentaltreatment of a hereditary illness: Wilson's dis-ease. Ann. intern Med. 1960, 53, 1151.

10. Holmberg, C. G., and Laurell, C.-B. Oxidase reac-tions in human plasma caused by coeruloplasmin.Scand. J. clin. Lab. Invest. 1951, 3, 103.

11. Dixon, W. J., and Massey, F. J. in Introduction toStatistical Analysis. New York, McGraw-Hill,1957, p. 209.

12. Gitlin, D., and Janeway, C. A. Genetic alterationsin plasma proteins of man in The Plasma Proteins,F. W. Putnam, Ed. New York, Academic Press,1960, vol. 2, p. 407.

13. De Vries, A., Rosenberg, T., Kochwa, S., and Boss,J. H. Precipitating antifibrinogen antibody ap-pearing after fibrinogen infusions in a patient withcongenital afibrinogenemia. Amer. J. Med. 1961,30, 486.

14. Gitlin, D., and Borges, W. H. Studies on the me-tabolism of fibrinogen in two patients with con-genital afibrinogenemia. Blood 1953, 8, 679.

15. Bennhold, H., and Kallee, E. Comparative studieson the half-life of I1`-labeled albumins and non-radioactive human serum albumin in a case ofanalbuminemia. J. clin. Invest. 1959, 38, 863.

16. Zilversmit, D. B. The design and analysis of iso-tope experiments. Amer. J. Med. 1960, 29, 832.

17. Sternlieb, I., Morell, A. G., and Scheinberg, I. H.Unpublished observations.

18. Sternlieb, I., Morell, A. G., Bauer, C. D., Combes,B., de Bobes-Sternberg, S., aud Scheinberg, I. H.Detection of the heterozygous carrier of the Wil-son's disease gene. J. clin. Invest. 1961, 40, 707.

19. Morell, A. G., and Scheinberg, I. H. Preparation ofan apoprotein from ceruloplasmin by reversibledissociation of copper. Science 1958, 127, 588.

20. Morell, A. G., and Scheinberg, I. H. Heterogeneityof human ceruloplasmin. Science 1960, 131, 930.

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