ABNORMALITIES OF COPPERMETABOLISMIN WILSON'SDISEASE ANDTHEIR RELATIONSHIP TO THE

AMINOACIDURIA

BY A. G. BEARNANDH G. KUNKEL

(From the Hospital of The Rockefeller Institute for Medical Research, New York, N. Y.)

(Submitted for publication September 2, 1953; accepted November 25, 1953)

In recent years an increasing amount of evidencehas accumulated suggesting that Wilson's diseaseis a metabolic disorder associated with disturbancesin copper and amino acid metabolism (1-8). Theurinary excretion of copper and amino acids arenow known to be increased in this disease (2, 3).The total plasma amino acids are not significantlyraised (4, 8), and the serum copper concentration,originally considered to be normal or increased,has recently been shown to be decreased (5). Areduced level of serum copper oxidase activity inWilson's disease has also been briefly reported (5).The findings of Holmberg and Laurell (9) indi-cated that oxidase activity in the serum is due toceruloplasmin the major copper containing proteinof serum. A deficiency of ceruloplasmin in Wil-son's disease, using spectrophotometric and im-munological techniques has also been demonstrated(7). A genetic analysis of a large group of pa-tients with Wilson's disease has been reportedseparately and indicates the disease to be inheritedin an autosomal recessive fashion (10).

The experiments to be described in this and acompanion paper ( 11) were undertaken to investi-gate further the abnormalities in copper and aminoacid metabolism in this disease and to correlatethese findings with the clinical condition of the pa-tient with emphasis upon the extent of hepatic andneurological involvement. A control group of pa-tients suffering from various types of cirrhosis ofthe liver was also studied.

MATERIAL AND METHODS

All patients studied were admitted to the hospital.Twenty-four-hour urine samples were collected in acid-cleaned vessels and stored at + 40C. Thymol crystals orchloroform (6 ml.) and toluene (6 ml.) were added aspreservatives. Urinary alpha amino nitrogen was deter-mined by the gasometric ninhydrin method of Van Slyke,MacFadyen, and Hamilton (12). The method of Brand,Harris, and Biloon (13) was used to detect qualitativelythe presence of urinary cystine and was also employed asa screening procedure in a search for aminoaciduria in

siblings of patients with Wilson's disease. Urinary glu-cose was estimated using the "Clinitest" 1 reagent, andvenous blood sugar according to the method of Nelson(14).

Serum and urinary copper were estimated in duplicateby a slight modification of the method described by Edenand Green (15). Serum copper estimations were carriedout in duplicate using 5 ml. samples; for urinary estima-tion of copper 15 or 20 ml. was normally used. The aver-age difference between duplicate analyses of a samplecontaining 5'Y of copper was 0.24 per cent. The averagedifference between duplicates can be calculated from theformula 2/1-S where a = S.D. When necessary, copperanalyses on 1 ml. samples of serum were carried out withconsiderable accuracy using the micro modification of thesame procedure (15).

Serum copper enzyme (ceruloplasmin) was estimatedby the method of Holmberg and Laurell (9) employingparaphenylendiamine (p.p.d.) as substrate. Oxygen up-take was measured in a Warburg flask at 370C. Thesolution of p.p.d. containing 5 mg. per ml. was broughtto pH 6.0 by the drop-wise addition of 1 N HG. The pHwas measured in a Beckman pH meter. One ml. of thep.p.d. solution together with 1 ml. of acetate buffer of pH6 was placed in the flask. Five-tenths ml. of serum,dialyzed at +4C. against acetate buffer (pH 6.0) fortwelve hours, was placed in the sidearm. On all occasions,a control sample using 0.5 ml. of distilled water was usedto correct for the oxygen uptake of paraphenylendiaminewhich occurred in the absence of ceruloplasmin.

Electrophoretic separation of sera was carried out in astarch-supporting medium by a previously describedmethod (16). Special precautions were taken to removetraces of copper by washing the starch with "Versene" 2and by doubly distilling the water used for the buffer solu-tion and the electrode vessels. The micro-copper proce-dure (15) was employed for analyses of the serum frac-tions displaced from the starch segments; 4 ml. of serumwere fractionated in each case.

CLINICAL MATERIAL

Seventeen patients with Wilson's disease were studiedand compared with 23 patients with cirrhosis and 12 nor-mal subjects.

'Ames Company, Inc., Elkhart, Indiana.2 The Versene used was kindly supplied by Riker

Laboratories, Inc., Los Angeles, California.

400

8 14 d| lM oW d o o

Sx!\}u 2 2 2 b o~~~~~~~~W) W) Go °o tW W

tv CIO 0. WUC U W

b.98§pitS| $ ° ^ § i ,, O jE s ^ ° , o ("

CO +++++ + ++ + + + + + + + + + 0 + + + + ++ + + + ~~~~~~~~~~~~~~~~~~~++

4i ~~+ + +a++m

E||+ + + + + + + + + + + +++, +o o ++ + + + + + 0 + + ++ + +_ _ _ ++ + + + +

+ + + + +

-gIg g > e'0 t0. o. .- 0 U

Z0 if 0 '0 ifom~~~ ~~~~ -_fo o o o-o

W 0 -0e0^Io4 0Ia#) aIoo " IN o IW WIN 0 1- 0l* - 0 0 0

No I * _ eqI - -- 1-

go+o o o o o + o+ ++ o o o

Izt 1 0 0 0 0 0 0° ° + 0 + 0 + 0 0 0

C83; Cs C CUC*]s

O 0

4t 0 I 0> e . e. u, -t bo - - -

~~~~~~~~~~~~;g~~~~~~S dEX;

*eZ~~~~a a a_

8~~~~£0 1o~ 0 £0 ; , a

C 0 0 0 +2

i~~~~~al .0E O u= =

V co co c 0o 0C0

0.4 | '0's w

~~I 4 - '0 0 if.~~~~ o- '0 0 0 0 C e 0 e'

. 6 . . ... b 0 00

ce 0 0 ~~~~~~~~00 co0 0.9~~~~~~~ 000 0

co '10 '

-II-X -I-X 0. --X-O -Q * X

0~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~0

4~~~~~~ ~~~~~~~~~~a('3.

(.3| > L .4X 3 3Q : > u5 I' >~T4

0) In 'IO &- 0o 0lk 0-0

401

C- -" -4 -u -'

0:

CI"

'UC.1

a5.

it

I

'14

I*bb

*e

---

13

4co0

la

tE0

.9

0A

0

.2

:F0

0

go0

0

a"

0

U

D

4-

01:

.2ba

to

I

I

A. G. BEARNAND H. G. KUNKEL

TABLE II

Comparison of serum copper, serum copper enzyme (ceruloplasmin) and urinary copper in normalsubjects, patients with cirrhosis of the liver and Wilson's disease *

Normals Cirrhosis Wilson's Disease(12] [23] [17]

Serum copper 108 ± 9.8 171 ± 47 60 ± 15.4,ug. /100 ml. [11] (20] [17]

Serum 'copper enzyme" 3.60 t 0.73 6.68 ± 2.39 0.90 ± 0.54,.uM. oxygen uptake/ [12) | 18] |15]

ml. /hr.

Urinary copper 48 t 16.3 157.4 ± 125.6 703 ± 318| ug./24 hrs. ) [18] |16]

(]No. of cases

*,Mean values with standardcdeviations are recorded.

The patients with Wilson's disease had varying degreesof hepatic and neurological involvement (Table I). Thepredominance, in the affected group, of males and of pa-

tients of Jewish and Italian extraction, has been discussedin a previous paper (10).

The patients with cirrhosis included 12 suffering fromnutritional LaEnnec's cirrhosis, 4 with biliary cirrhosis,and a group of 7 young girls with severe liver disease ofuncertain etiology which has previously been described(17).

RESULTS

Serum copper

The serum copper levels found in normal sub-jects, in patients with Wilson's disease and in pa-

tients suffering from cirrhosis of the liver are

shown in Table II. It will be seen that the mean

copper concentration in the serum of patients withWilson's disease was considerably lower than thatfound in normal subjects, although in one patientthe serum copper concentration was in the normalrange." The wide scatter shown for the values ob-tained for the patients with cirrhosis may be a re-

sult of the heterogeneity of the clinical material.The mean serum copper concentration in the threevarieties of cirrhosis studied is shown in Table III.The patients with nutritional LaEnnec's cirrhosisdid not show a serum copper concentration greatlyin excess of normal, while those with biliary cir-rhosis had high levels. Patients with cirrhosis of

8 An additional case has since been studied and a normalserum copper concentration found.

the liver of unknown etiology occupied an inter-mediate position.

Serum copper enzyme

The serum copper enzyme (ceruloplasmin) wasmeasured in the three groups studied and the re-sults are illustrated in Table II. A striking de-crease in oxidase activity was noted in all patientswith Wilson's disease, while an increase in activitywas noted in patients with cirrhosis of the liver.Table III illustrates the results obtained when thepatients with cirrhosis of the liver are further clas-sified. The greatest activity was again found tooccur in the patients with biliary cirrhosis. Thosewith nutritional cirrhosis had an oxidase activityonly slightly greater than normal, and those pa-tients with cirrhosis of unknown etiology again oc-cupied an intermediate position.

A parallelism between the serum copper concen-tration and the oxidase activity was first shown byHolmberg and Laurell (9). Figure 1 illustratesthe parallelism in normal subjects, patients withcirrhosis of the liver and patients with Wilson'sdisease. The intercept of the regression line drawnto fit the experimental points suggests that in se-rum some copper is present which is not bound asceruloplasmin and hence is not enzymatically ac-tive. In patients with Wilson's disease the cerulo-plasmin activity appears to be somewhat less thanwould be expected from a consideration of the se-

402

ABNORMALITIES OF COPPERMETABOLISM IN WILSONS DISEASE

TABLE III

Comparison of serum copper, serum copper enzyme and urinary copper invarious types of cirrhosis of the liver

*UndeterminedLaennec etiology Bilary

Serum copper 142 4 8.0 163 4t 29.1 245 4 18.9pg./100 ml. [8) [8] [4]

Serum "copper enzyme" 5.46 4 2.14 6.22 i 1.46 9.45 1 2.781uM oxygen uptake/ml./hr. [6] [8] [4]

Urinary copper 86.2 4 48.4 174.7 4 62.8 269pg./24 hrs. [9] [7] 565

[2]

[)No. of cases.i Cirrhosis of the liver, occurring chiefly in young girls.

rum copper concentration, indicating that the non-ceruloplasmin copper may be relatively increased.

In an attempt to evaluate the significance of theenzyme activity measured above and to study thedistribution of copper among the various serumproteins, analyses of copper and enzyme activitywere carried out on fractions of serum separatedelectrophoretically in a starch supporting medium.Difficulties were encountered because of the ina-bility to prevent completely interference by tracesof contaminating copper. In each experiment morecopper was found on adding up the values obtainedfor each electrophoretic segment than was presentin the original serum. Most of the contaminatingcopper became attached to the albumin fraction, afact that was apparent in repeated experimentswith the same serum. The height of the albuminpeak was reduced under conditions where extreme

14 0- X C1rrmhosis

12.0 - * Miary irrho=s* Normalso Wilsons d&sease

10.0-

8.0 -

# Xauptake/

0 40 80 120 160 200 240 280Serum copper (,u9./100 ml.)

FIG. 1. THE RELATIONSHIP BETWEEN THE SERUMCOPPER CONCENTRATIONAND SERUM COPPER ENZYME(CERULOPLASMIN)

care was exerted to exclude contaminating copper.To accomplish this end purified human albuminwas applied to the starch block in front of the se-rum and this albumin picked up copper while thealbumin in the serum became less contaminated.Figure 2 illustrates typical results obtained froma large number of sera of the three types tested forcopper distribution. The curve obtained for pro-tein in the same electrophoretic segments used forthe copper analyses is illustrated for the normalserum. Two major copper peaks are visible, afront peak (dotted) representing the copper at thealbumin, and a middle peak in the a2 globulinregion. Determination of oxidase activity in thevarious fractions showed that all the activity wasassociated with the a2 globulin copper peak, whichtherefore represents ceruloplasmin. In some ex-periments a blue band against the white starchbackground could be seen in this region. The se-rum from the patient with Wilson's disease showedalmost complete absence of the a2 globulin copperpeak. In the case of cirrhosis, on the other hand,both this peak and the oxidase activity of thewhole serum, were higher than normal.

The significance of the other small peaks isuncertain because of difficulty in defining ex-actly the baseline. The height of the a2 globu-lin copper peak showed very little variationfrom one experiment to another on the same se-rum in contrast to the variability of the copperpeak in the albumin fraction. In addition, recoveryexperiments indicated that close to the total copperof the serum from normal individuals and patientswith cirrhosis of the liver could be accounted forin this peak. Whether any of the copper found in

403

A. G. BEARN AND H. G. KUNKEL

the albumin fraction was non-contaminating cop-per was not clear from these experiments. In thesera of patients with Wilson's disease, the recoveryexperiments indicated that the low a2 globulincopper peak accounted for less of the total serumcopper than in normal subjects and. patients withcirrhosis of the liver. Studies with radioactivecopper have shown final localization of radioactivityin the a2 globulin fraction after oral administrationin normal individuals. The latter results confirmthe localization of copper in this fraction found inthe direct determinations of copper in the electro-phoretic fractions.

Urinary copper

Urinary excretion of copper was greatly in-creased in all patients with Wilson's disease andwas slightly increased in most patients with cir-rhosis of the liver. These results are illustrated inTables II and III. Although the highest valueswere obtained from patients with Wilson's disease,some overlap occurred with patients suffering frombiliary cirrhosis. Under standard dietary condi-tions, day-to-day fluctuations in the urinary ex-

0.4- Cirrhosis0.5

0.2 ..

0.1 A

*0.4- Wi1son'sDiseaseCh 023-'h

.20CL0.11~ ~ ~ ~ I 0.Normal § l 0.6

0.4 \ 0.5

p I~~~~~~~~~~~~~0

1 5 10 15Fraction No.

FIG. 2. CURVES SHOWINGTHE DISTRIBUTION OF COP-PER IN VARIOUS SERUMFAcTIONs SEPARATEDBY ZONEELECTROPHORESIS

The protein pattern is illustrated for the normal serum.The ceruloplasmin peak is in fraction 10. The broken linelargely represents contaminating copper in the albuminfractions.

0~

C.

U

B

oDD -C~

1 5 JoDays

i5 20

FIG. 3. THE EFFEcT OF DIETARY COPPERINTAKE ONTHE URINARY EXCRETION OF COPPER IN WILSON'SDIsEAsE

cretion of copper in all groups studied were foundto be relatively small and to be independent ofurinary volume.

Amino acidsUrinary excretion of alpha amino nitrogen was

increased in 16 of the 17 cases of Wilson's dis-ease. On a fixed protein diet, urinary excretionof alpha amino nitrogen of individual patients withWilson's disease showed little day-to-day fluctua-tions, and was independent of urinary volume.Random samples of urine from 35 unaffectedsiblings of patients with Wilson's disease wereexamined and no significant aminoaciduria wasdetected in any of the cases studied, nor wasconsistent aminoaciduria found in patients withcirrhosis of the liver. Patients in hepatic comawere not tested.

GlycosuriaGlycosuria was found in 5 of the 17 patients with

Wilson's disease and in these patients some corre-lation was noted between urinary glucose and uri-nary amino acid excretion. In addition, a rise inthe amino acid excretion was often accompaniedby an increased urinary excretion of glucose. Infour of the five cases blood sugar determinationswere carried out and showed that the fasting bloodsugar was not elevated.

Spectrographic analysis 4

Spectrographic analysis of the urine was carriedout in one patient with Wilson's disease and one

4 Spectrographic analysis was carried out through thecourtesy of Dr. R 0. Roblin, Jr., American CyanamidCompany.

404-

ABNORMALITIES OF COPPERMETABOLISMIN WILSONS DISEASE40

Days

FIG. 4. THE EFFECT OF VARIATIONS IN THE DIETARYPROTEIN ON THE URINARY EXCRETION OF COPPER AND

AMINO Acms IN A PATIENT (L. A.) WITH WILSON'S

DISEASE

normal subject to study the excretion of othertrace metals in Wilson's disease. Only the ex-

cretion of copper was found to be increased. Thelevels of iron, cobalt, nickel, and zinc were withinnormal limits.

Relation of urinary copper and amino acid excre-

tion to dietary intake of copper and protein

Urinary excretion of copper in normal subjectsand in patients with Wilson's disease was largelyindependent of copper intake. Thus, increasingthe intake of copper from 0.5 mg. per day to 12mg. per day did not result in a significant increase

tr By, ;op

IVC. MO.

ZJ.P-~*10000

Day

Keoo

a M2400

N~~~~~~~~~~200~ ~ ~ ~ ~

100

1 5 10 15

Days

FIG. 5. CHANGESIN THEURINARYCOPPERANDAMINOACID EXCRETION FOLLOWINGCHANGESIN DIETARY PRO-TEIN IN PATIENT (C. P.) WITH WILSON'S DISEASE

.,J2

co260 :@ J'4\

1 5 10 15 20 25Days

FIG. 6. EFFECT OF DIETARY PROTEIN ON URINARYEXCRETION OF COPPERAND AMINO ACIDS IN A NORMALSUBJECT

in the urinary excretion of copper (Figure 3).This result was found whether copper in the dietwas increased or came from added copper sulfate.In patients with Wilson's disease an increasedprotein intake resulted in a prompt and parallelincrease in the urinary excretion of copper andamino acids. The changes produced in two dif-ferent patients, are illustrated in Figures 4 and 5.In normal subjects increasing the dietary proteincaused no detectable rise in the urinary excretionof copper. A typical response is shown in Fig-ure 6.

The excess amino acids excreted in patientswith Wilson's disease placed on a high protein dietwere greater than occurred in normal subjects onthe same diet. Both groups were placed on a 200Gm. protein diet and the total increased daily ex-cretion of amino nitrogen in Wilson's disease wasapproximately three times more than occurred innormal subjects.

Effect of cortisone, "Benemid",i and mercurialdiuretics

The relationship between urinary copper andurinary amino acids in Wilson's disease was fur-ther demonstrated by the administration of corti-sone. The increase in the amino acid excretion inWilson's disease which followed the administrationof cortisone was again promptly accompanied bya parallel increase in the urinary excretion of cop-per (Figure 7).

Tp-[di-n-propylsulphamyl]-benzoic acid.

405

A. G. BEARN AND H. G. KUNKEL

Days

FIG. 7. EFFEcT OF CORTISONEON URINARY ExCRETIONOF COPPERAND AMINO AcIDs IN A PATIENT WITH WIL-SONS DISEASE

Mercurial diuretics and "Benemid" administeredin full doses to two patients were not found to in-fluence the excretion of amino acids in Wilson'sdisease significantly. Mercurial diuretics had noeffect on the urinary excretion of copper. Fol-lowing the administration of Benemid, however,there appeared to be a slight increase in the uri-nary excretion of copper in one patient.

Effect of chelating agents

The administration of BAL increased the uri-nary excretion of copper in all patients to whomitwas given (Table IV). This was related to theamount of BAL given since increasing the doseincreased the urinary excretion of copper.

TABLE IV *The effect of various substances on urinary excretion of

copper in Wilson's disease

MeanNo. of increase

Compound Dose patients per 24 hr.

BAL 2.5 mg./Kg. 9 51

Versene 1 Gm. 1 67(Intravenously) 2 Gm. 3 138

Versene 30 mg./Kg. 3 5(Orally)

Cortisone 100 mg. 3 31200 mg. 3 65

* Figures represent mean values obtained during periodof maximum effect.

"Versene" (20 per cent solution of calcium di-sodium salt of ethylenediaminetetracetic acid in500 ml. of normal saline) was administered intra-venously to patients with Wilson's disease. In allcases an increase in the urinary excretion of cop-per was noted and the mean results are shown inTable IV. Increasing the amount of "Versene"administered again increased the urinary excre-tion of copper. "Versene" administered orally(Table IV) did not increase the urinary excretionof copper significantly in the doses used. Thedaily urinary excretion of alpha amino nitrogenwas not altered by the administration of BAL or"Versene".

The increased urinary excretion following theadministration of BAL or intravenous "Versene"was independent 'of the degree of hepatic orneurological involvement.

DISCUSSION

The low serum copper concentration in 16 6 of17 patients with Wilson's disease confirms earlierwork from this laboratory (5). Recently, Lahey,Gubler, Cartwright, and Wintrobe (18) have re-ported three additional patients with Wilson's dis-ease who had a low concentration of copper in theserum. In patients with cirrhosis of the liver, onthe other hand, the serum copper is frequentlyelevated. It has been shown by van Ravesteynthat copper is normally excreted in the bile (19).This observation is in agreement with the presentfinding that the degree of elevation of the serumand urinary copper in patients with cirrhosis isdue at least in part to the degree of biliary obstruc-tion associated with the cirrhotic process. Pa-tients with classical nutritional LaEnnec's cirrhosiswithout significant biliary obstruction have normalor slightly elevated serum copper and urinary cop-per concentrations while the higher values en-countered in both serum and urine were those oc-curring in patients with biliary cirrhosis.

The greater part of the circulating serum cop-per is bound to a protein exhibiting oxidase ac-tivity. This protein has been called ceruloplasminby Holmberg and Laurell (9). Ceruloplasminhas recently been measured by Scheinberg andGitlin (7) using specific immunochemical tech-niques and also spectrophotometrically. They re-

6 See footnote 3.

406

ABNORMALITIES OF COPPERMETABOLISM IN WILSONS DISEASE

ported eight patients with Wilson's disease inwhom the ceruloplasmin concentration was con-siderably lower than normal and this finding is inaccord with the results of the present study.Holmberg and Laurell (9) were the first to sug-gest that there was a parallelism between the con-centration of copper in the serum and the oxidaseactivity. Further light on the- relation of serumcopper to various serum proteins has been derivedfrom the experiments using zone electrophoresis.The major copper peak in normal serum and inthe serum from patients with cirrhosis appeared inthe a, globulin region. The latter peak was mark-edly depressed in the sera of patients with Wilson'sdisease. The electrophoretic experiments alsoconfirmed the specificity of the oxidase measure-ments of whole serum as an index of its cerulo-plasmin level. Copper bound to other proteins inserum gave no enzymatic activity with the sub-strates employed.

The present work does not suggest that the maincopper protein in serum (ceruloplasmin) functionsas a transport mechanism in serum in a mannersimilar to the iron binding protein. This possi-bility was also considered unlikely by Gubler,Lahey, Cartwright, and Wintrobe (20) who haveshown that following the oral ingestion of copperthere is no change in the "indirect reacting" cop-per. It is probable that "indirect reacting" copperrepresents ceruloplasmin. The possibility of anenzymatic function in the body must be considered,particularly in view of the broad distribution ofcopper enzymes in tissues and the activity ex-hibited by this protein with synthetic substrates.Whether a deficiency of ceruloplasmin representsthe primary genetically determined abnormalityin Wilson's disease remains to be determined.

Preliminary observations suggested that therewas a parallelism between the excretion of copperand the excretion of amino acids in patients withWilson's disease (5). This observation was sup-ported and amplified by the recent findings ofMatthews, Milne, and Bell (8) who showed thatingestion of amino acids led to a prompt rise inthe urinary excretion of copper and by the pres-ent work where increases in dietary protein gaverise to an increased excretion of copper and aminoacids in the urine. In normal subjects, despitean increase in the urinary alpha amino nitrogen,there was no detectable increase in the urinary

copper excretion. In Wilson's disease as a resultof a high protein diet an increased absorption ofamino acids will occur and some of the excessamino acids may combine directly with the hightissue copper and an increase in the urinary excre-tion of copper will result. In normal subjects inwhom tissue copper is not raised and in whomtherenal threshold is not lowered no significant in-crease in the urinary excretion of copper will occur.

Although the association between urinary copperand amino acid excretion has been demonstratedthere is no evidence that the copper and the aminoacids are excreted as copper amino acid complexes,moreover, there are many other substances ex-creted in the urine other than amino acids to whichcopper could become bound. An attempt was madeto determine the substances in the urine to whichthe copper was bound by zone electrophoresis ex-periments following the oral administration ofradioactive copper. Several copper peaks were ob-tained when the experiment was run at the pH ofthe excreted urine but whether the copper wasbound to certain specific amino acids or peptideswas not entirely clear.

It has been suggested that in Wilson's dis-ease there is a lowered renal threshold for aminoacids and that this abnormality accounts for thefinding of an increased excretion of amino acidsin the urine (4, 8). The present finding that,following the. administration of a high proteindiet to patients with Wilson's disease, a greaterquantity of amino acids was excreted in the urinethan occurred in normal subjects could also becited to support this hypothesis. However, con-sideration of the behaviour of individual aminoacids, which is discussed more fully in the follow-ing paper (11), suggests that such a hypothesismay not explain all of the experimental findings.

The increased excretion of copper followingcortisone was expected since it is known that cor-tisone increases the urinary excretion of aminoacids (21). Mercurial diuretics and "Benemid,"substances well known to have an action on therenal tubules, did not alter amino acid excretionand hence urinary copper excretion was not sig-nificantly increased. The increased urinary ex-cretion of copper following the administrationof BAL, or "Versene" intravenously was not as-sociated with an increase in amino acid excretion,probably because both BAL and "Versene" are

407

A. G. BEARN AND H. G. KUNKlEL

themselves substances which form a chelate withcopper which is readily excreted in the urine.

"Versene" administered orally did not resultin an increase in the urinary excretion of copperin patients with Wilson's disease. This observa-tion is in contrast to recent findings in the treat-ment of lead poisoning (22) where an increasedurinary excretion of lead followed the administra-tion of "Versene" orally. The dose employed inthe treatment of lead poisoning, however, wastwice that employed in the present study.

Van Ravesteyn (19) has suggested that in nor-mal subjects urinary copper excretion does notreflect dietary intake of copper. This study fur-nishes further evidence for this concept, since bothnormal subjects and patients with Wilson's dis-ease, provided they were placed on a constant pro-tein intake, did not show a significant increase inthe urinary excretion of copper despite very largevariations in copper in the diet. However, fol-lowing the intravenous administration of traceamounts of radioactive copper it has been possibleto demonstrate an increased urinary excretion ofcopper and a decreased fecal excretion comparedwith normal subjects.

There was no clear cut relation between theduration or severity of the disease process in pa-tients with Wilson's disease and the degree of ab-normality found in copper and amino acid metabo-lism. These biochemical findings were similarlyunrelated to the degree of hepatic or neurologicalinvolvement or the presence of Kayser-Fleischerrings. The clinical improvement which sometimesappeared to follow the administration of variouschelating agents was not related to the amountof copper excreted; the latter was similar in alltypes of the disease whether or not subsequent im-provement occurred.

The finding that Wilson's disease is inheritedin an autosomal recessive fashion (10) suggestedthe possibility that asymptomatic siblings mightpossess a biochemical abnormality. Uzman andHood (23) have reported a family with Wilson'sdisease in whomexamination of four asymptomaticsiblings revealed an aminoaciduria. However,this family, in view of the large number of bio-chemically affected individuals described, is geneti-cally atypical. It is also noteworthy that examina-tion of 35 siblings in the present study failed toreveal an aminoaciduria in any individual. How-

ever, an increased amino acid excretion was notfound in one patient with undoubted Wilson's dis-ease (11) and this observation suggests that anaminoaciduria may not be an early manifestationof the disease, and examination of asymptomaticsiblings for abnormalities in copper metabolismshould be preferentially undertaken. Investigationof the copper metabolism in some of the asympto-matic siblings has thus far failed to reveal anyabnormalities.

As a hypothesis it is suggested that in Wilson'sdisease as a result of an increased absorption ofcopper this metal accumulates in the tissues andappears in the urine. If the deposition of copperin the liver becomes excessive cirrhosis may de-velop and, in an analogous fashion, accumulation ofcopper in the kidney may give rise to renal tubulardamage. If this occurs an increased urinary ex-cretion of amino acids, peptides and occasionallyglucose will result.

SUMMARY

1. Copper and amino acid levels in serum andurine were determined in 17 patients with Wilson'sdisease and compared with a control group of pa-tients with cirrhosis of the liver as well as withnormal individuals.

2. Reduced copper levels in the serum and ele-vated urinary concentrations of copper were char-acteristic findings of the group with Wilson'sdisease. The presence of both abnormalitiesproved more characteristic of the disease thaneither one alone.

3. Markedly reduced levels of the serum copperprotein, ceruloplasmin, were found in all cases ofWilson's disease, and represented the most specificbiochemical abnormality found. The specificity ofthe enzymatic method for the determination ofceruloplasmin was demonstrated by procedures ofzone electrophoresis.

4. A parallelism was demonstrated between theurinary copper and amino acid excretion in Wil-son's disease. Increasing the amino acid excre-tion by increasing the protein intake, or by theadministration of cortisone resulted in a parallelincrease in the urinary excretion of copper.

ACKNOWLEDGMENTS

It is a pleasure to acknowledge our indebtedness toDoctor Houston Merritt, Doctor Theodore J. C. von

408

ABNORMALITIES OF COPPERMETABOLISM IN WILSONS DISEASE

Storch, Doctor Harold Wolff, and Doctor Morris Benderfor having so kindly referred patients to us.

Wewish to thank Mr. Charles Galati for expert tech-nical assistance.

REFERENCES

1. Glazebrook, A. J., Wilson's disease. Edinburgh M. J.,1945, 52, 83.

2. Uzman, L., and Denny-Brown, D., Amino-aciduriain hepato-lenticular degeneration (Wilson's dis-ease). Am. J. Med. Sc., 1948, 215, 599.

3. Denny-Brown, D., and Porter, H., The effect of BAL2,3-dimercaptopropanol) on hepatolenticular de-generation (Wilson's disease). New England J.Med., 1951, 245, 917.

4. Cooper, A. M., Eckhardt, R. D., Faloon, W. W., andDavidson, C. S., Investigation of the aminoaciduriain Wilson's disease (hepatolenticular degeneration):Demonstration of a defect in renal function. J.Clin. Invest., 1950, 29, 265.

5. Beam, A. G., and Kunkel, H. G., Biochemical abnor-malities in Wilson's disease. J. Clin. Invest., 1952,31, 616.

6. Moore, S., and Stein, W. H., Aminoaciduria in Wil-son's disease. International Congress of Biochem-istry, 2nd. Resumes des communications, Paris,Lons-le-Saunier, M. Declume, 1952, p. 367.

7. Scheinberg, I. H., and Gitlin, D., Deficiency of ceru-loplasmin in patients with hepatolenticular degenera-tion (Wilson's disease). Science, 1952, 116, 484.

8. Matthews, W. B., Milne, M. D., and Bell, M., Themetabolic disorder in hepato-lenticular degenera-tion. Quart. J. Med., 1952, 21, 425.

9. Holmberg, C. G., and Laurell, C.-B., Oxidase reac-tions in human plasma caused by coeruloplasmin.Scandinav. J. Clin. & Lab. Invest., 1951, 3, 103.

10. Beam, A. G., Genetic and biochemical aspects of Wil-son's disease. Am. J. Med., 1953, 15, 442.

11. Stein, W. H., Beam, A. G., and Moore, S., Theamino acid content of the blood and urine in Wil-son's disease. J. Clin. Invest., 1954, 33, 410.

12. Van Slyke, D. D., MacFadyen, D. A., and Hamilton,P. B., The gasometric determination of amino acidsin urine by the ninhydrincarbon dioxide method.J. Biol. Chem., 1943, 150, 251.

13. Brand, E., Harris, M. M., and Biloon, S., Cystinuria.The excretion of a cystine complex which decom-poses in the urine with the liberation of free cystine.J. Biol. Chem., 1930, 86, 315.

14. Nelson, N., A photometric adaptation of the Somogyimethod for the determination of glucose. J. Biol.Chem., 1944, 153, 375.

15. Eden, A., and Green, H. H., Micro-determination ofcopper in biological material. Biochem. J., 1940,34, 1202.

16. Kunkel, H. G., and Slater, R. J., Zone electrophoresisin a starch supporting medium. Proc. Soc. Exper.Biol. & Med., 1952, 80, 42.

17. Kunkel, H. G., Ahrens, E. H., Jr., Eisenmenger, W. J.,Bongiovanni, A. M., and Slater, R. J., Extremehyperganmmaglobulinemnia in young woman withliver disease of unknown etiology. J. Clin. Invest.,1951, 30, 654.

18. Lahey, M. E., Gubler, C. J., Cartwright, G. E., andWintrobe, M. M., Studies on copper metabolism.VII. Blood copper in pregnancy and various pa-thologic states. J. Clin. Invest., 1953, 32, 329.

19. van Ravesteyn, A. H., Metabolism of copper in man.Acta med. Scandinav., 1944, 118, 163.

20. Gubler, C. J., Lahey, M. E., Cartwright, G. E., andWintrobe, M. M., Studies on copper metabolism.IX. The transportation of copper in blood. J. Gin.Invest., 1953, 32, 405.

21. Bardawill, C. J., Gornall, A. G., Nishikawara, M., andWightman, K. J. R., Studies on the behaviour ofurinary amino nitrogen, serum alkaline phosphatase,and pseudo cholinesterase activity during ACTHtherapy. Proc. Second Clinical ACTHConference,J. R. Mote, ed., The Blakiston Co., Phila., 1951, 1,264.

22. Sidbury, J. B., Jr., Bynum, J. C., and Fetz, L. L., Ef-fect of chelating agent on urinary lead excretion.Comparison of oral and intravenous administration.Proc. Soc. Exper. Biol. & Med., 1953, 82, 226.

23. Uzman, L. L., and Hood, B., The familial nature ofthe amino-aciduria of Wilson's disease (hepato-lenticular degeneration). Am. J. Med. Sc., 1952,223, 392.

409