Life Sciences Vol. 16, pp . 1025-1032 Printed in the II .S .A .
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) .
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
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
ml red cells (24) .
The superoxide dismutase content of the
red cells is expressed as units (see below) per ml red cells .
and assay are all done on the same day. Superoxide Dismutase Assay. tract containing 1 .8 to of Lowry et al .
mg protein per ml as determined by the method
using crystalline bovine serum albumin as the standard .
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 .
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
One unit of superoxide dismutase activity inhibits the
rate of cytochrome c reduction by of
per min. at
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
This was determined by the method of Cox (28) . Results
The erythroq~te superoxide dismutase and serum ceruloplasmin levels from
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
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
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)
M. C .
G . B.
Overall Mean (tSD)
31 .8 (±6.0)
J. R .
M. C .
G. C .
F. C .
F. C .
31 .5 (±12 .8)
*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 .
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 .
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-
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)
Cerulo lasmin ** m~g~âT~-
Hepxtic P. V .
K. V .
*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
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 .
expert, dedicated assistance of Mr . James F . Jennings is gratefully acknowledged . References 1.
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