Life Sciences Vol. 16, pp . 1025-1032 Printed in the II .S .A .

Pergamon Prese

SUPEROXIDE DISMUTASE ACTIVITY (ERYTHROCUPREIN) IN WILSON'S DISEASE Nicholas M. Alexanders and Cordon D. Benson 2 sDivision of Clinical Pathology, Departinent of Pathology School of Medicine, University of California, San Diego, California 92103 and 2Department of Medicine, Jefferson Medical College of Thomas Jefferson University, Philadelphia, Pennsylvania 19107 (Received in final form February 18, 1975)

Surmnary Erythroçyte superoxide dlsmutase (erythrocuprein) levels were determined in cells from normal subjects and from patients with Wilson's disease. The concentration of this copper-containing enzyme was essentially identical in both groups, even though serum cer~uloplasmin was markedly reduced, or absent, in Wilson's disease, The observed concentration of the dismutase confirms previous results by others using immunochemical procedures . Extended therapy with D-penicillamine resulted in a 25 to 43% decrease in superoxide dismutase activity, and an 81 to 99% decrease in serum ceruloplasmin. Our results indicate that erythrocuprein levels are independent of serum cervloplasmin concentration. Wilson's disease (hepatolenticular degeneration) is an inherited, autosomal, recessive disorder that is characterized by two cardinal biochemical features : 1) excessive tissue copper deposition, and usually 2) a marked diminution in ceruloplasmin, the serum copper binding glycoprotein (1-8) . It has been postulated that the disease is due to deficient hepatic ceruloplasmin synthesis and decreased biliary excretion of copper (9,10) .

Con-

sequently, copper accumulates in some tissues because it cannot be adequately sequestered by serum ceruloplasmin .

Another hypothesis contends that cop-

per accumulation is due to abnormally increased binding by tissue proteins such as metallothionein (11) .

However, several criticisms have cast doubt

on both hypotheses (6-8,12) . 1025

Superoxide Diamutaae ead Wilson'e DieeaBe

1026

Vol. 16, No . 7

Ceruloplasmin oxidizes (13-16) many biologically important substances (Few, eptnephrine, etc.) .

Although this property is probably related to

iron utilization in vivo (17-19) and is important for serum ceruloplasmtn assays, its role in the etiology of Wilson's disease is not clear. Some evidence with leucocytes from Wilsonian patients suggests that cervloplasmin functions in the transport of copper to essential cellular enzymes, such as cytochrome oxidase (20) .

The data on copper transport into

erythrocytes is conflicting, since although there is a reduced rate of radiocapper incorporation into erythrocuprein of red cells from patients with Wilson's disease, the total flux of copper into the cells is initially increased (21) .

Moreover, erythrocuprein concentration in red cells from

normal and Wilsonian patients are identical, as measured by quantitative immunochemical techniques (22,23) .

A possible explanation for these divert

gent results is that free apoerythrocuprein (21) exists in red cells and is detected by the immunochemical procedures . To study this possibility, we measured erythrocuprein levels in red cells from normal and Wilsonian patients by utilizing its superoxide dismutase activity (24-26) .

This enzymatic property (02 + 2H+ -~ H202)

allows one to detect intact erythrocuprein because the apoprotein is inactive .l Materials and Methods Chemicals . 10962 .

Xanthine was purchased from Schwarz/Mann, Orangeburg, N .J .

Cytochrane c, type III, and milk xanthine oxidase, grade II were

products of Sigma Chemical Company, St . Louis, Mo . 63178.

All other chemicals

were of the highest possible purity from commercial sources . Preparation of Red Cell Extract.

Erythrocytes were collected from

heparinized blood, washed three times with 0.9~ NaCI, hemolyzed and fractionated with chloroform-ethanol (Tsuchihashi method) as described by McCord and Fridovich (24) .

Complete extraction of superoxide dismutase is achieved

by this method and yields a clear, colorless extract in which 1 ml is equilA preliminary report of this work appeared in Fed . Proc . 31, 697 (1972) .

Superoxide Diamutasa and Wilson's Disease

Vol . 16, No . 7

valent to

0.64

ml red cells (24) .

102 7

The superoxide dismutase content of the

red cells is expressed as units (see below) per ml red cells .

Extraction

and assay are all done on the same day. Superoxide Dismutase Assay. tract containing 1 .8 to of Lowry et al .

2.9

This assay

(24)

mg protein per ml as determined by the method

using crystalline bovine serum albumin as the standard .

(27)

The reaction mixture in a volume of 3 ml contains M EDTA,

is performed with ex-

5 x 10 -5 M

M ferricytochrome c, 10 ul extract and

5 x 10 -5

potassium phosphate buffer .

0.05

xanthine, 10 -4 M, pH 7.8

c is started

The reduction of ferricytochrome

by stirring in 10 ul of a xanthine oxidase solution containing and

units/mg .

1 .6

0 .0125

(22°-23°)

mg/m1

One unit of superoxide dismutase activity inhibits the

rate of cytochrome c reduction by of

9 .5

per min. at

550

nm .

50%

and is equal to an absorbante change

The assays are performed at room temperature

in a Gilford model 2000 recording spectrophotometer, and are the

mean of duplicate analyses with a coefficient of variation not greater than

9% .

Cer~uloplasmin .

This was determined by the method of Cox (28) . Results

The erythroq~te superoxide dismutase and serum ceruloplasmin levels from

6

male and

9

female normal subjects, ranging in age fran 10 to 40 years,

are listed in Table 1 .

Although there are individual variations, the over-

all mean concentrations of superoxide dismutase are essentially identical in both groups, and there are no apparent differences with respect to age. Other experiments not shown in the table indicated that the enzymatic activity is destroyed by heating the extract at 100° for 5 minutes .

The serum ceru-

loplasmin levels also vary between individuals, but the overall mean concentration is the same in the two groups .

The concentration of erythrocyte

superoxide dismutase is not correlated with serum ceruloplasmin levels ; for example, female subject G. C . (age

16)

has 317 units of superoxide dis-

mutase and 17 .0 mg/dl serum ceruloplasmin, whereas, nearly the same concen-

Superoxida Dismutase and Wilson's Disease

1028

Vol . 16, No . 7

TABLE 1 Erythrocyte Superoxide Dismutase and Serum Ceruloplasmin Levels in Normal Controls Superoxide Dismutase e ells) (Units m

~C~er~~ulo ?Jasmin* (mg/dl)

Males

Acme

P. C.

10

329

37 .5

E. C.

12

305

32 .3

M. C .

13

268

38 .6

J. T.

30

259

32 .7

R. B.

31

297

24 .1

G . B.

40

277

25 .3

Overall Mean (tSD)

289 (t26)

31 .8 (±6.0)

J. R .

14

251

27 .1

M. C .

15

253

22 .3

G. C .

16

317

17 .0

F. C .

18

291

58.3

V. S.

21

309

36 .2

S. S.

24

256

28 .5

M. B.

25

280

22 .2

F. C .

40

359

43 .4

L.W .

40

252

28 .2

285 (f38)

31 .5 (±12 .8)

Females

Overall Mean

(±SD)

*Normal range is 20-45 mg/dl tration of dismutase is found with twice as much serum ceruloplasmln (female Y. S ., age 21) . Erythrocyte superoxide dismutase and serum ceruloplasmin wncentrations in three patients with Wilson's disease are presented in Table 2. P. V . and K. V. are sisters .

P. V . presented with an hemolytic anemia and

Superoxide Diemutase and Wilson's Disease

Vol. 16, No . 7

liver disease, whereas, K. V, was asymptomatic .

Patient C . K, presented

with neurological manifestations of Wilson's disease .

The initial

levels of superoxide dismutase in all three patients are nearly identical to the controls in Table 1, while the serum ceruloplasmin is markedly reduced (P . V . and K. V.) or absent (C . K.) .

The concentration of super-

oxide dismutase in the red cells declined 43X and 25X after 2 years of therapy with D-penicillamine in subjects P . V . and K. V. respectively, and 38X after 64 weeks of therapy in C . K.

Serum ceruloplasmin levels also

decreased by 99~ and 81~ after penicillamlne therapy in patients P . V . and K. V. respectively . Discussion Previous analyses of serum ceruloplasmin, erythrocyte copper and erythrocuprein levels (immunochemical) indicated that the amount of erythrocuprein in red cells was independent of the ceruloplasmin concen tration (22) .

Our results support this interpretation, since the super-

oxide dismutase activity in red cell extracts from patients with Wilson's disease does not significantly differ from normal controls .

Indeed, normal

levels of erythrocyte superoxide dismutase are maintained even when there is no measurable amount of ceruloplasmin in serum (patient C . K., Table 2) . This implies that copper for erythropoiesis is probably derived fr~am serum albumin (29,30) or other freely diffusible copper complexes . Our data do not exclude the possibility that some cells obtain copper by catabolizing serum ceruloplasmin .

This mechanism might account for the

correlation between serum ceruloplasmin levels and the concentration of cytochrame oxidase in 1eu wcytes (20), which potentially possess this type of catabolic activity . Our data indicate that hunan erythrocytes contain 9-10 mg superoxide dismutase per 100 ml, based on a specific activity of about 3,000 units/mg at 22°-23° (24) .

This value is in excellent agreement with the results

obtained by immunochemical methods that employed pure erythrocuprein anti-

1029

1030

Superoxide Dismutase and Wilson's Disease

Vol . 16, No . 7

TABLE 2 Erythroçyte Superoxide Dismutase and Serum Ceruloplasmin Levels in Patients with Wilson's Disease CNS C. K .

Sex A e

D-Penicillamine Thera * ee s~

Superoxide Dismutase (Units m e ells)

F/22

0

263

0

24

276

0

36

276

0

64

163

0

0

288

7.7

2

266

6.4

3

261

5. 7

9

263

7.1

35

226

1 .7

l16

l63

0 .1

0

251

3.6

2

238

3 .4

28

276

1 .4

106

188

0.1

Cerulo lasmin ** m~g~âT~-

Hepxtic P. V .

K. V .

F/14

F/13

*Received 250 mg, 3 or 4 times daily **Normal range is 20-45 mg/dl gen (23) .

Even though copper turnover in erythrocuprein proceeds at a

faster rate in normal red cells than in cells of patients with Wilson's disease (21), the concentration of erythrocuprein in both cells is essentially identical .

These findings are not entirely surprising, inasmuch as no pro-

tein turnover occurs in the red cell .

In addition, it is unlikely that

significant amounts of free apocytocuprein exists in red cells because

Vol. 16, No . 7

Superoaide Diemutaee and Wilson's Disease

1031

there is a 2 1/2 fold excess of intracellular, freely diffusible copper iftat is capable of donating copper to erythrocuprein (21,23) . Acknowledgement This work was partially supported by USPHS Grant AM-15778 .

The

expert, dedicated assistance of Mr . James F . Jennings is gratefully acknowledged . References 1.

S . A . K . WILSON, Brain 34 295-509 (1912) .

2.

A . G, BÉARN, Am, J . Med . 15 442-449 (1953),

3.

I . H, SCHEINBERG and A . G . MORELL, J . Clin . Invest . 36 1193-1201 (1957) .

4.

G . E, CARTWRIGHT, R . E . HODGES, C . J . GUBLER, J, P, MAHONEY, K . DAUM, M . M, WINTROBE, and W . B . BEAN, J . Clin . Invest, 33 1487-1501 (1954). .

5.

C . J, EARL, M . J . MOULTON, and 8 . SELVERSTONE, Am . J, M~d . 17 205213 (1954) .

6.

A . G . BÉARN in The Metabolic Basis of Inherited Disease, J . B . Stanbury, J . B . Wyn aarden, D, S . Fredrickson, Eds . pp . 1033-1055 (McGraw-Hill, N .Y . 1972,

7.

I . H . SCHEINBERG and I . STERNLIEB, Ann . Rev . Med . 16 119-134 (1965) .

8.

G . W . EVANS, Physiol, Rev . 53 535-570 (1973) .

9.

S . O'REILLY, P . M . WEBER, M, OSWALD, and L . SHIPLEY, Arch . Neurot . 25 28-32 (1971) .

10 .

D . J . FROMMER, Gut 15 125-129 (1974),

11 .

G, W . EVANS, R, S . DUBOIS and K . M . HAMBIDGE, Science 181 1175-1176 (7973),

12 .

I, H . SCHEINBERG, Science 185 1184 (1974) criticizes the work in ref, 11 and a response by Evans appears in Science 185 1185 ; a critical summary of ref . 11 is also presented by J . L . Boyer, Gastroenterology 66 470 (1974) .

13 .

G . CURZON in Wilson's Disease : Some Current Concepts, J . M . Walshe and J . N . Comings, Eds . p . 96 (Thomas, Springfield, I11 . 1961),

14 .

S . OSAKI, D . A . JOHNSON, and E . FRIEDEN, J . Biol . Chem, 241 2746-2751 (1966) .

15 .

G . A, JAMIESON, J, Biol . Chem . 240 2019-2027 (1965) .

16 .

S . AKERFELDT, Science 125 117-118 (1957) .

17 .

H . P, ROESER, G . R . LEE, S . NACHT, and G . E . CARTWRIGHT, J . Clin . Invest . 49 2408-2417 (1970),

18.

G . M, BRITTIN, and Q . T . CHEE, J . Lab . Clin . Med . 74 53-59 (1969) .

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Superozide Dismutasa and Wilson's Disease

Vol . 16, No . 7

19 .

E . FRIEDEN, in Trace Element Metabolism in Animals, W. G. Hoekstra, J . W . Suttie, H . E. Ganther and W. Mertz, Eds . p. 105-118 (University Park Press, Baltimore, Md . 1974) .

20 .

M . H. K. SHOKEI R and D. C. SHREFFLER, Proc . Nat. Acad . Sciences 62 867-872 (1969) .

21 .

P. Z . NEUMANN and M. SILVERBERG, Nature 213 775-779 (1967) .

22 .

G. S . SHIELDS, H . MARKOWITZ, W. H. KLASSEN, G. E . CARTWRIGHT and M. M . WINTROBE, J . Clin . Invest . 40 2007-2015 (1961) .

23 .

M. J . STANSELL and H. F. DEUTSCH, Clin . Chim . Acta 14 598-607 (1966) .

24 .

J. M . McCORD and I . FRIDOVICH, J . Biol . Chem . 244 6049-6055 (1969) .

25 .

J. M. McCORD and I . FRIDOVICH, J . Biol . Chem . 244 6056-6063 (1969) .

26 .

I . FRIDOVICH, Accts . Chem . Res . 5 321-326 (1972) .

27 .

0. H. LOWRY, N . J. ROSEBROUGH, A . L . FARR and R. J. RANDALL, J . Bio1 . Chem . 193 265-275 (1951) .

28 .

D. W. COX, J . Lab . Clin . Med. 68 893-904 (1966) .

29 .

S . J. ADELSTEIN and B. L . VALLEE, New Eng. J . Med. 265 892-897 (1961) .

30 .

J . A. BUSH, J. P . MAHONEY, C. J . GUBLER, G. E . CARTWRIGHT, and M. M. WINTROBE, J. Lab . Clin . Med. 47 898-906 (1956) .

Superoxide dismutase activity (erythrocuprein) in Wilson's disease.

Life Sciences Vol. 16, pp . 1025-1032 Printed in the II .S .A . Pergamon Prese SUPEROXIDE DISMUTASE ACTIVITY (ERYTHROCUPREIN) IN WILSON'S DISEASE Ni...
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