Copper has been recognized for many years as anessential nutrient (1 ) . If it is assumed that the norma! adult is in copper balance (i.e., that his totalquantity of body copper does not increase or decrease significantly with time), then it is reasonableto infer that the amount of copper absorbed by himmust equal the amount excreted (2) . Urinary excretion of this metal is usually negligible—10—60@g/day (2). Maintenance of an average total-bodycopper content of 100 mg (3—5)must therefore represent a balance between absorption and excretionin the gastrointestinal tract.

Metabolic balance studies have been greatly simplified by using a whole-body counter capable ofdetecting small amounts of gamma-emitting nuclidesin vivo. Nevertheless, variations in data resultingfrom urinary excretion of a small fraction of absorbed copper and from elimination in the stool of alarger fraction of orally absorbed copper togetherwith the limitation placed upon observations of wholebody and excreta by the short physical half-life ofMCu ( 12.8 hr) have made necessary the development of a fairly complex model for the presentinquiry. Comparisons are reported of copper absorption and gastrointestinal excretion in seven normal subjects, in two patients with Wilson's disease(6) and in one patient with an undiagnosed disorder of copper metabolism.

SUBJECTS AND METHODS

Subjects. Ten subjects were studied. The sevencontrols were asymptomatic volunteers in whom noabnormality believed to affect copper metabolismwas found. They were patients who had been admitted to the hospital for treatment of conditionsunrelated to the subject of this inquiry and withoutcirrhosis or abnormalities of liver function. Of thethree patients with disease pertinent to this inquiry,two had Wilson's disease and were receiving penicillamine. The third, a 16-year-old male, had an undiagnosed abnormality of copper metabolism thatwas not Wilson's disease.

Throughout the investigation, all of the 10 subjects were maintained on a constant metabolic dietin the Clinical Study Center of the San FranciscoGeneral Hospital. One complete absorption studywas performed in each of five control subjects; threestudies were performed in one control and fourstudies in one. One complete study was performedin each of the three patients.

Method. To each fasting subject °5Zr,as carrierfree zirconium oxalate, was given orally in dosesvarying from 0.5 to 10.0@ This was followedimmediately by the oral administration of 100—500

@@Ciof 64Cu as copper acetate with specific activityof 0.3—1.3,@Ci/,@gcopper.

Counting was performed in a low-backgroundwhole-body counter with 2-ft-thick concrete wallslined interiorly with @/8-in. lead. An 11.9 X 4-in.Na!(Tl) crystal was placed at the center of the arcformed by a counting couch with a 1-meter radiusof curvature ( 1-meter arc geometry) . The outputof the crystal was analyzed by a 400-channel analyzer(RIDL Model 34-1 2 B). The 64Cu and 95Zr werecounted in 400—600-keV and 650—850-keV windows, respectively. Appropriate corrections weremade for Compton events of zirconium appearing inthe copper window.

In each subject, counting was performed dailyfor 4 days beginning with the administration of theoral dose; this is the maximum possible countingtime in view of the 12.8-hr half-life of °4CuThebolus of orally administered radioactive copper wasfollowed through the gastrointestinal tract by scintiphotographs obtained sequentially with an Angerscintillation camera (Nuclear-Chicago). All urine andstools were collected and counted daily in the wholebody counter under appropriate geometric control,by calculations based on comparison with appropriatestandards similarly counted.

Received June 21, 1968; revision accepted March 7, 1969.For reprints contact: Paul Weber, The Clinical Study

Center, San Francisco General Hospital, 22nd and PotreroAve., San Francisco, Calif. 94110.

Volume 10, Number 9 591

GASTROINTESTINAL ABSORPTION OF COPPER:

STUDIES WITH 64Cu, 95Zr, A WHOLE-BODY COUNTER

AND THE SCINTILLATION CAMERA

Paul M. Weber, Sean O'Reilly, Myron Pollycove and Leroy Shipley

San Francisco General Hospital, University of California School of Medicine,San Francisco, California

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WEBER, O'REILLY, POLLYCOVE AND SHIPLEY

One week after administration of the oral dose,0.5—10 @@Ciof °@Zroxalate was given again to eachfasting subject, this time followed immediately bythe intravenous administration of 500 @@Ciof °4Cuwhich had been incubated with 10—20ml of thesubject's plasma for 30 mm at 37 °Cto insure proteinbinding of the copper. Thereafter, each of the 10subjects was again counted in the whole-bodycounter, daily for 4 days. All urine and stools wereagain collected and counted daily and related toappropriate standards.

The net dose (oral or intravenous) was determined by applying to the administered dose the ratioobtained by relating the whole-body counting rateof an intravenous dose of 64Cu to the counting rateof the syringe counted on the 1-meter arc, correctedfor counts remaining in the syringe after injection. Inpreparatory studies we had determined this ratio atfrequent intervals up to the first 4 hr and had foundit to be most constant immediately after injection

(0.73 ±0.035 in 12 normal subjects). Since at thistime hepatic extraction has not yet begun, all of theradiocopper must be within the vascular space sothat this factor must represent self-absorption bythe subject. Plasma radioactivity was measured ina standard well gamma-ray counter using a 400—600-keV window.

The model and the calculation method for netcopper absorption are shown schematically in Fig. 1.Copper absorption, A, is equal to the ratio of thenet retention of radiocopper in the whole body afteroral administration of copper, R@,to the net reten

tion of radiocopper in the whole body after intravenous administration of copper, 1-E1@,where E@ isthe fractional excretion of radiocopper into the gastrointestinal tract after intravenous administration:

Excretory data for both R@and E1@are incompletebecause of the short half-life of °4Cu.Both were corrected for this incompleteness on the basis of thedata obtained from the zirconium which had beenadministered orally as a nonabsorbed stool marker(7) as follows:

R = 1 —D0-Ru@,and E = D1@-Ru1@(See Appendix).0D0 Iv iv

D0 and D1@are the net oral and intravenous dose,respectively; Ru@and Ru1@represent the copper retamed in the whole body, uncorrected for stoolrecovery; S@and Si@are the fractions of zirconiumrecovered in stool during the oral and intravenousphase of the study, respectively. Both@ and Ru1@are corrected for counts recovered in the urineduring the course of the study although this fractionwas trivial except in subjects receiving penicillamine.This calculation presumes that °5Zris excreted atthe same rate as unabsorbed gastrointestinally cxcreted °4Cu.This presumption has been validatedexperimentally when more than 25 % of zirconiumoxalate was recovered in stool.

This technique is valid only if three additionalassumptions are correct: ( 1) That copper absorptionis equal to net copper retention plus the fractionexcreted after absorption. This is self-evident fromthe definition of absorption. (2) That the excretedfraction of the absorbed oral fraction is equal to thefraction excreted after the intravenously administered dose. Studies related to the validity of this assumption have been in progress in our laboratoryfor some time, and present data suggest that it is correct. Externally placed probes and whole-body scanning techniques have shown that approximately the

A —@-@-- same fraction of orally absorbed and intravenously

I- Ejv injected copper localize in the liver. Further support

is lent to this assumption by the similarity of plasmaradioactivity curves after oral and intravenous copper administration, and we are now collecting dataon the labeled copper content of bile which we hopewill be definitive in this regard (such studies havebeen made feasible only within the past year bythe availability of °TCu). Our results suggest thenthat the hepatoenteric metabolism of copper differsqualitatively from that of other metals such as iron.(3 ) That the fractional excretion after intravenousinjection is constant from week to week. This as

64Cu 95Zr

FIG. 1. Schematicmodelfor calculationof netcopperabsorption (A). R. is net retention of radiocopper after oral administration and@ is fractional excretion of radiocopper into gastrointestinal tract after intravenous administration.

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SubjectFractionof oralFraction

excretedafterintravenousNo.

Age Sexdoseabsorbedadministration(yr)(%)(•i')

Doseof carrier CuadministeredSubjectsFraction

of ‘CuabsorbedMeanRange(mg)(No)(%)(To)

GASTROINTESTINAL ABSORPTION OF COPPER

that absorption of copper, unlike that of other dcments such as iron, is not limited. In the two subjects with Wilson's disease who were receiving penicillamine, almost all orally administered radiocopperwas absorbed while gastrointestinal excretion of theintravenously administered dose was within the norma! range.

The amount of carrier copper given with the oraldose, related to the fractional absorption of radiocopper during the oral phase, is shown for normalsubjects in Table 2. The quantities of carrier givenvaried over a 1,000-fold range to span the range ofcopper generally presented to the gastrointestinaltract at one time (estimated to be 1.5—5.0mg ofcopper per meal) . Five groups are defihed accordingto the quantity of carrier administered. While theaverage absorption values suggest that the fractionof radiocopper absorbed decreased as the quantityof carrier increased, the ranges of absorption areextremely wide within each group and they overlapmarkedly. In addition, the subject with the highestpercentage of absorption was the one who had beengiven the largest amount of carrier; when this subject was studied by varying the amounts of carrierpresented with the oral dose, fractional absorptionsshowed no direct relationship to those amounts.

When all data were plotted with fractional absorption on the x-axis and amount of carrier copper

TABLE 1. SUMMARY OF COPPER ABSORPTIONAND GASTROINTESTINALEXCRETION

Controls:119M7523229M350343M75

378619421F4320528M71<1660M6314766M15

8797331

14Mean

59.811.5Patients8 19 M 87 149* 41 F 100 19

lot 16 M 74 24

0 Wilson's disease; taking penicillamine.

t Undiagnosedabnormalityof copper metabolism;nottaking penicillamine.

TABLE 2. CARRIER COPPER ADMINISTERED,RELATED TO FRACTION OF RADIOACTIVECOPPER ABSORBED,IN SEVEN NORMALSUBJECTSGROUPED BY CARRIER DOSE

.

A

. A

.

I . .

.

. CONTROLSUBJECTS

A WILSONS-PENICILLAMINE

I I I I@

<0.10.1—0.90.9—1.65.110.1

3 66 37—877 58 15—863 49 35-631 33 —1 â€9̃7 —

10.0

6

4

2

E@ 1.0

E@ 6

‘U@ 4

U

‘Ua.a.0@

U 6

4

2

0.0@- 20 40 60 80 100

% ABSORPTION

FIG. 2. Relationship between copper absorption and carriercopper, latter on a logarithmic scale.

sumption is relevant because the studies are performed sequentially. This variable was controlled aswell as possible by maintaining the patients in theclinical study center on metabolic diets.

RESULTS

Net absorption of orally administered radiocopperand net excretion of the intravenously administerednuclide are shown in Table 1. In the normal subjects, net absorption of the orally administered dosevaried from 15 to 97% with a mean of approximately 60% . The fraction excreted after intravenousadministration varied through a much narrowerrange—0—23%with a mean of 11.5% . This widevariation in the fraction absorbed.by control subjectssuggests that the normal range is very broad and

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along a logarithmic scale on the y-axis (Fig. 2), nosignificant relationship between these two variablesemerged.

Plasma copper radioactivity is plotted againsttime after oral administration of radioactive copperin Fig. 3. Absorption was maximal in the first hourbut continued for several hours. Scintiphotographs

of the abdomen show that at 1 hr, the time of maximal absorption, the radiocopper was in the stomachand duodenum; at 2 hr, in the small intestine. At5 hr, when the bolus of radioactivity was largely inthe ileocecal region and first portion of the largebowel, absorption was probably completed.

Figure 4 relates the plasma radioactivity curvesafter intravenous administration to those after oraladministration of radiocopper, the oral curve beingnormalized with respect to the intravenous data asto time. During plasma clearance of the intravenouslyadministered isotope, feedback to plasma began at

about 25 mm, too early to permit determination ofthe total length of the period of absorption of theoral dose; however, the plateau reached by the oralcurve about an hour before there is a plateau of theintravenous curve indicates that some of the orallyadministered copper was still being absorbed, up to3½hr after it had been received.

Data before and after administration of penicillamine were available in three control subjects. Theeffect of penicillamine on their gastrointestinal cxcretion of intravenously administered copper and ontheir net absorption of orally administered copper isshown in Table 3. In Subjects 3 and 6 gastrointestinalexcretion diminished after the administration ofpenicillamine. In Subject 5 gastrointestinal excretion was insignificant initially and did not change inresponse to penicillamine. The percent of copperabsorbed after penicillamine is available only forSubjects 5 and 6; in both, there was a modest decrease from pre-penicillamine values. The primaryeffect of penicillamine was a marked increase in theurinary excretion of copper.

Fraction of °‘CuFraction of°@CuabsorbedexcretedinstoolWith

WithoutWithWithoutpenicil-penicil penicil- penicil

Subjectlamine laminelaminelomine(No)(%)(‘1°)(‘to)(%)37519

10

Y2 1 2 3 4 5

TIME (HOURS)

WEBER, O'REILLY, POLLYCOVE AND SHIPLEY

‘I)

-aa-z

‘0

U

E

a.U

6

FIG. 3. Plasmacopperradioactivityrelatedto timeafter oraladministration. Scintiphotographs of abdomen at 1, 2 and 5 hrare superimposed on curve and show activity in stomach andduodenum when adsorption is maximal.

E

a.U

5@

64CU IN PLASMA

FIG. 4. Relationshipbetweenplasmaradioactivitycurvesfollowing intravenous and oral administration of radiocopper withoral curve normalized as to time relative to the intravenous data.

III

TABLE 3. EFFECTOF PENICILLAMINE ONABSORPTION AND GASTROINTESTINAL

EXCRETION OF °4CuIN 3 NORMAL SUBJECTS

1 2 3 4 5 6

TIME (HOURS) 8637

5 716 63

53, <1 149 14 4

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GASTROINTESTINAL ABSORPTION OF COPPER

primarily upon pool size and turnover rate. Evaluation of these data requires the longer observationperiods now possible with ‘7Cu.One would expectthat over a sufficient interval, equality of copperabsorption and excretion should be demonstrable.

The observations reported here do not lend themselves to interpretation relative to mechanisms involved in the positive copper balance known toexist in Wilson's disease (1 1—13), largely becausesubjects were limited in number and were studiedonly while under therapeutic control by penicillamine. Patient 10, without Wilson's disease, is unusual. When measured on several occasions byoxidative and spectrophotometric methods, ceruloplasmin was found low; but when measured immunochemically, it was found quantitatively normal. Finaldiagnosis has not been made; but his copper absorption and excretion data do not differ significantlyfrom those of the control subjects reported.

The three control subjects studied before andafter penicillamine excreted less °4Cuinto the stoolwhile taking this drug than when not taking it. Thisis in contrast to our observation in a previous metabolic balance study (14) that penicillamine appearedto increase gastrointestinal excretion of copper inpatients with Wilson's disease. The normal range ofgastrointestinal excretion of intravenously administered radiocopper during penicillamine administration in both of the Wilsonian homozygotes in thepresent study may be interpreted as support for thisfinding for it is virtually certain that without penicillamine their gastrointestinal excretion of copperwould have been significantly less. Further studiesare needed on the effect of penicillamine on the gastrointestinal excretion of copper in Wilson's diseaseand on the apparent decrease in net copper absorption in response to penicillamine in two normal subjects found here.

CONCLUSION

A technique for measuring copper absorption hasbeen described in which 64Cu is administered twice.The first dose is given orally. One week later, thesecond dose is administered intravenously. Simultaneously with each dose, 95Zr oxalate is given orallyas a nonabsorbable stool marker. Data so obtainedindicate a wide range of absorption values in normalsubjects and absence of a well-defined carrier effect.After intravenous administration of copper, an average of 11.5% is excreted into the gastrointestinaltract (range 0—23%).

In the normal subject, copper absorption is maxima! during the first hour while the metal is in thestomach and duodenum; absorption continues for atleast 3.5 hr.

DISCUSSIONThe availability of only a single radioactive iso

tope of copper (°4Cu)has precluded studies of themetabolism of this metal by methods such as thedouble-isotope technique used by Fawwaz and coworkers (8) in investigating the disposition of iron.With the very recent availability of the nuclide 67Cu,we are beginning to explore the application of multiple-isotope procedures to the physiologic handlingof copper. The studies with °4Cu(half-life 12.8 hr)reported here were designed to offset the limitationsof the single isotope in providing new informationon gastrointestinal absorption and excretion in man.

Our findings are somewhat at variance with someof the admittedly limited data from studies in manreported by others. Beam and Kunkel (9) reportedup to 33 % stool recovery of intravenously administered °4Cu,figures higher than any found by us.Bush and coworkers (10) found an average of12.4% of intravenously administered °4Cuin thestool of normal subjects, the highest value being16. 1%—close to our normal values. Beam and Kunkel (9) reported recovery of 26% of orally administered 64Cu in the stool over 4 days; while Bushand his group (10) recovered up to 95% of anoral dose in the stool over a similar period.

The wide variations in net absorption in our normal subjects was most surprising. While at variancewith the findings of the investigators just cited, theyare not inconsistent with the broad discrepancybetween the results reported separately by them.These observations suggest that the control mechanism regulating copper absorption is much morelabile than that regulating the absorption of, forexample, iron. Such lability might be expected, asrequisite to homeostasis of the small total-body poolof copper (in the 100-mg range) , in contrast to thetotal-body pooi of iron (probably close to 4,000mg) . Since the fractional excretion of copper varieswithin relatively narrow limits, the findings suggestthat variation in absorption is a primary controlmechanism.

The other possible interpretation of these data isthat the subjects were not in a steady state eventhough they were hospitalized in a metabolic ward.Variations in copper absorption noted in repeatedstudies of the same subjects suggest that this mightbe the case. Perhaps even a metabolically controlleddiet contains elements or compounds that can limitor enhance copper absorption by such mechanismsas copper binding or even by competition for absorption sites.

The high absorption levels in our normal subjectsdo not imply positive copper balance, for once radiocopper is absorbed, its fractional excretion depends

595Volume 10, Number 9

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WEBER, O'REILLY, POLLYCOVE AND SHIPLEY

11. BICKEL, H., NEALE, F. C. AND HALL, G. : A clinicaland biochemical study of hepato-lenticular degeneration(Wilson's disease). Quart. I. Med. 26:527, 1957.

12. SUNDERMAN, F. W., JR., WHITE, J. C. AND SUNDERMAN, F. W. : Metabolic balance studies in hepatolenticular

degeneration located with diethyldithiocarbamate. Am. I.Med. 34:875, 1963.

13. GOLDSTEIN, N. P., RANDALL, R. V., GROSS, J. B.AND McGucIuN, W. F. : Copper balance studies in Wilson's

disease : observations on the effect of penicillamine, carbacrylamine resins, and potassium sulfide. Arch. Neurol.12:456, 1965.

14. O'REILLY, S. AND BANK, W. : Treatment of Wilson'sdisease—a new oral chelating agent. Nature 212:1,597,1966.

Urinary excretion of copper is negligible exceptduring the administration of penicillamine.

In a limited number of control subjects, penicillamine tended to decrease both net absorption (orretention) of orally administered copper and fractional gastrointestinal excretion.

Two patients with Wilson's disease, while takingpenicillamine, showed nearly maximal absorptionbut “normal―gastrointestinal excretion of copper.

ACKNOWLEDGMENT

Supported in part by Grant NB-07102-01 from the National Institute of Neurological Diseases and Blindness,National Institutes of Health, Bethesda, Maryland. Thestudies were carried out in the General Clinical ResearchCenter, FR-83, at San Francisc' General Hospital, supportedby Division of Research Grants and Facilities, NationalInstitutes of Health.

REFERENCES

1. ELVEHJEM, C. A.: The biological significance of copper and its relation to iron metabolism. Physiol. Rev. 15:471, 1935.

2. CARTWRIGHT,G. E. AND WINTROBE,M. M. : Coppermetabolism in normal subjects. Am. I. Clin. Nutr. 14:224,1964.

3. Sm@iN, W. H. : AAAS Symposium on GeochemicalEvolution. Table I. Denver, 1961.

4. Cisou, TiJNO-PI ANDADOLPH,W. H. : Copper metabolism in man. Biochem. I. 29:476, 1935.

5. GUBLER, C. J. : Copper metabolism in man : report toCouncil on Foods and Nutrition. I. Am. Med. Assoc. 161:530, 1956.

6. O'REILLY, S. : Problems in Wilson's disease. Neurology 17:137, 1967.

7. MACDOUGALL,L. D. : Estimation of fat absorptionfrom random stool specimens:measurementby zirconium95 and iodine-131. Am. I. Diseases Children 108:139,1964.

8. FAWWAZ, R. A., WINCHELL, H. S., POLLYCOVE, M.,SARGENT, T., ANGER, H. AND LAWRENCE, J. H. : Intestinaliron absorption studies using iron-52 and Anger positroncamera. J. Nuci. Med. 7 :569, 1966.

9. Br.@ar.i,A. G. @NiKUNKEL,H. G. : Metabolic studiesin Wilson's disease using Cu-64. I. Lab. Clin. Med. 4@:832,1955.

10. BUSH, I. A., MAHONEY, J. P., M@.RKowrrz, H.,GUBLER, C. J., CARTWRIGHT, G. E. AND WINTROBE, M. M.:Studies on copper metabolism. XVI. Radioactive copperstudies in normal subjects and in patients with hepatolenticular degeneration.I. Clin. invest. 34: 1,766, 1955.

APPENDIXD0@ Net oral and intravenous dose, respectively, inD1@5 cpm, obtained by multiplying net counts

injected by 0.73 as described.

Ru0@ Radiocopper retained in the whole body inRuj@J cpm uncorrected for completeness of stool

recovery after oral and intravenous dose,respectively. Data obtained either fromwhole-body counter or from stools.

S@@ Fractions of zirconium recovered in stool dur@ J ing the oral and intravenous phase of the

study, respectively.

Fractional net retention of MCu in the wholebody after orally administered copper corrected for completeness of stool recovery.

R0 —1 D0-Ru0 _@ D0-Ru@,— — SO/DO@@ S0D0

E1,, Fractional excretion of °4Cuin stool after i.v.administration of copper corrected forcompleteness of stool recovery.

E1, = D1@—Ru,@= D1@RU1@

lv! iv lv iv

A Net fractional absorption of °4Cu.

V

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1969;10:591-596.J Nucl Med.   Paul M. Weber, Sean O'Reilly, Myron Pollycove and Leroy Shipley  and the Scintillation Camera

Zr, a Whole-Body Counter95Cu, 64Gastrointestinal Absorption of Copper: Studies with

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