TRACE METAL PATTERNS IN DISEASE STATES An altered pattern

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TRACE METAL PATTERNS IN DISEASE STATES An altered pattern
TRACE METAL PATTERNS IN DISEASE STATES
II.
COPPER STORAGE DISEASES, WITH CONSIDERATION OF JUVENILE
CIRRHOSIS, WILSON'S DISEASE, AND HEPATIC COPPER OF THE
NEWBORN
E . M. B U T T , M . D . , R. E . NUSBAUM, P H . D . , T. C. G I L M O U R , B.A., AND S. L. D I D I O , B.A.
Department
of Pathology, Los Angeles County Hospital, and University
Medicine, Los Angeles,
California
of Southern California
School of
An altered pattern of trace metals in dis- levels of silver are found in other organs
eases accompanied by an excessive storage with increased copper storage. High levels of
of hepatic iron has been described in a pre- copper and silver in juvenile and Laennec's
vious paper.2 The pattern consisted of a cirrhoses are found only in the liver. This
significant increase of lead, molybdenum, organ dissimilarity is important in the biocalcium, and copper, and a decrease of chemical differentiation of Wilson's disease
aluminum. Similar patterns were reported and the other 2 afflictions.
in rats on decreased or substantial protein
Reduced hepatic levels of molybdenum,
diets augmented with iron and in rabbits on zinc, and iron are found in juvenile and
diets containing iron. While copper was in- Laennec's cirrhoses.15 Similar reductions of
creased in iron storage conditions in human metal levels are found in Wilson's disease,
and animal livers, its quantitative relation however, with profound alterations of the
to iron was less consistent than that of lead, liver and evidence of hepatic failure.
molybdenum, or calcium.
An interesting geographic variation of the
This report is concerned with an altera- metal pattern of Wilson's disease is observed
tion of the metal pattern in several organs in material from New York City: lead levels
incident to increases of copper. The condi- are found to be elevated, as well as those of
tions studied are juvenile cirrhosis, hepato- copper and silver.
lenticular degeneration (Wilson's disease),
METHOD
Laennec's cirrhosis, and excessive hepatic
copper storage in premature infants.
The analytic work was accomplished with
Hepatic copper levels are found to be a spectrochemical method, using a 13^-m.
elevated in juvenile cirrhosis, Wilson's dis- grating "spectrograph (with photographic
ease, and Laennec's cirrhosis. Except for recording) for the earlier work, and a revariations associated with severe damage of cently acquired direct reading Industrial
the liver, the hepatic metal pattern in copper Research Quantometer* for some of the
storage diseases is increased in silver, with later work. The quantometer and its use will
little change in other metals. The levels of be described in a paper to be published in
silver parallel roughly those of copper. Analytical Chemistry.9 The procedures for
Moreover, in Wilson's disease elevated preparing the tissue samples, introducing
them into the electrical discharge and exReceived, July 23, 1958; accepted for publica- citing the spectrums were the same as
tion August 18.
previously described.1
D r . B u t t is Chief Pathologist, Los Angeles
Sections of the tissues for microscopic
County Hospital; Dr. Nusbaum is Physicist in
charge of Spectrographic Analysis, Atomic Energy study were stained with hemotoxylin and
Project, University of California; Mr. Gilmour is eosin, and special stains of sections of the
Chief Spectrographic Technician, and Mr. DiDio liver were made for identification of copper
is Assistant Spectrographic Technician, Univer- and iron pigments. Rubeanic acid stain was
sity of Southern California School of Medicine.
This work was aided by grant No. H683 (7)
from the National H e a r t I n s t i t u t e , U. S. Public
H e a l t h Service, Bethesda, Maryland.
479
* Applied Research Laboratories, Glendale, California.
480
B U T T ET
used for localization of copper13 and Perls'
reaction for detection of iron.
RESULTS
Juvenile Cirrhosis
(Including all types of hepatic cirrhosis
under 15 yr.)
Enormous quantities of hepatic copper
were found but the brain levels were within
normal range. Seven cases in our files had
an average hepatic copper value of 92.4 mg.f
ranging from 177.0 to 39.0 mg. (Table 1,
Fig. 1). Three died within 21 days of birth;
their average hepatic copper was 52.3 mg.,
approximately the same as in Wilson's disease. The remaining 4 cases, ages 5 to 15,
had an average hepatic copper of 122.6 mg.
Case 461. A 7-year-old Negro boy entered
the hospital on November 25, 1951, complaining of hematemesis. The past history
was uneventful except for an unusual number of epistaxes. Death, 3 days later, was
caused by rupture of esophageal varices
with gastrointestinal hemorrhage, secondary
to postnecrotic cirrhosis. The weight of the
liver was 850 Gm., spleen 475 Gm., and
brain 1260 Gm. The liver was coarsely
nodular. Histologically, broad bands of partially hyalinized and vascularized fibrous
tissue were observed, enclosing small and
large islands of polygonal cells, infiltrated by
moderate numbers of lymphocytes and
polymorphonuclear leukocytes. There was
moderate bile duct hyperplasia. The cells
were free of fat. Sections of the brain revealed no evidence of basilar nuclear degeneration. Copper levels were: liver 51.0
mg., kidney 3.1 mg., brain 5.9 mg. Iron
levels were: liver 12.8 mg., kidney 8.1 mg.,
and brain 5.5 mg. The liver was free of iron
pigment. Rubeanic stain of the liver revealed a moderate amount of copper pigment, rather coarsely clumped in polygonal
cells, chiefly around the peripheries of the
lobules (Fig. 2). No copper was visible in
the spleen.
Case 778. A Caucasian girl, age 17, had
cirrhosis of the liver for at least 10 years,
and died of anemia and hepatic failure. Dist All figures refer to mg. per 100 Gm. of dry
tissue.
AL.
Vol. 80
tribution of iron pigment was similar to
that in hemochromatosis. The pancreas was
fibrotic and dark brown. The liver was
coarsely nodular and microscopically not
unlike case 461, except for the presence of
large areas of necrosis and the presence of
pigment in the polygonal cells. The cirrhosis
seemed to be of the postnecrotic type, and
no doubt preceded the deposition of iron in
the tissues. Stainable iron was found in the
stomach, duodenum, adrenal gland, thyroid, pituitary, spleen, kidneys, and heart.
Inasmuch as the disease had existed without
anemia for so many years, it was more
convenient to classify the case as one of
hemochromatosis developing in a juvenile
nodular cirrhosis. The most amazing feature
was the extremely heavy storage of copper—
100 mg. (Fig. 3). While the copper was
slightly increased in the brain (6 mg.), the
storage was much less than that seen in
Wilson's disease. Basal ganglion degeneration was not noted grossly or microscopically.
Case 843. This 21-day-old male Caucasian,
a feeding problem since birth, developed a
persistent but mild diarrhea 3 days prior to
admission. The mother was Rh negative,
baby Rh positive, and the Coombs test negative. The hemoglobin was more than 18 Gm.,
white blood count 26,800, nonprotein nitrogen 87. He developed postprandial vomiting,
with severe diarrhea, and died on August 22,
1953. At autopsy, the sclerae were faintly
icteric. The 310-Gm. brain was free of gross
or microscopic alterations. The 95-Gm.
liver had a coarsely nodular surface. The
extrahepatic bile ducts were patent. Microscopically, the liver revealed a moderate
increase of periportal fibrous tissue, enclosing large and small islands of polygonal cells.
Bile duct hyperplasia was moderate. No
giant cells were seen. Copper was located
chiefly in the cells of the peripheries of the
lobules, for the most part finely divided, but
in some areas coarsely granular; it was not
found in the Kupfer cells. The appearance
of the copper was not unlike that seen in
case 601 (Fig. 4). Iron was 240 mg. and
copper 39 mg. Iron pigment was also found
in the spleen and lymph nodes. The copper
level of the brain was 1.75 mg.
Bee. 1958
481
COPPER STORAGE DISEASES. II
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482
BUTT ET
A.
B.
C
D.
Vol. 30
AL.
JUVENILE CIRRHOSIS
HEPATOLENTICULAR DISEASE
(WILSON)
LAENNEC CIRRHOSIS
CONTROL-AVERAGE ALL DISEASES
AV. = AVERAGE
D*NUMBER.OF CASES
AB.
C.
D.
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1,
LIVER.
BRAIN
LIVER
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LIVER Q L I V E R AV. 4.SJ t aS89
BRAIN [J BRAIN /W. 3.-12 t 0.209
O10
2030-10
Mq. PER.
B
10.26
B
50«)'I0809O:ia>10203O4O50M>1O8090200
100 qms. DRY TISSUE
FIG. 1. Storage of copper in the liver and brain
Case 915. The Rh-positive mother of this
full term, 3-day-old Negro boy had no recorded maternal complications. At autopsy,
the infant was fairly well developed and
nourished, the sclerae slightly yellow. The
brain, studied by Dr. C. B. Courville, neuropathologist, was said to reveal severe cerebral congestion, patchy nerve cell loss, and
focal areas of necrosis of the cortex. The
right lobe of the 42-Gm. liver was nodular,
the left less involved (Fig. 5). The histologic pattern was greatly disturbed, owing
to necrosis and collapse of the fibrous tissue
framework and slight proliferation of fibrous
tissue involving the periportal regions and
extending into the lobule, associated with a
moderate infiltration of lymphocytes, monocytes, and a few polyhuclear leukocytes.
Bile duct hyperplasia was minimal. Many
giant cells of hepatic cell origin were present.
Special stains revealed that there was much
iron pigment in Kupfer, giant, and liver cells,
as well as in the connective tissue. The
rubeanic acid stain revealed coarse granules
of copper pigment in the giant, Kupfer, and
liver cells. Histiocytes in the collapsed
fibrous framework also contained copper
(Figs. 6, 7, and 8). Smaller amounts of
copper pigment were found in the less disturbed liver cells. Iron, but no copper pigment was present in the thymus, kidney
tubules, and acinar cells of the pancreas.
Hepatic iron was 352 mg., copper 74 mg.,
and silver 0.16 mg. Brain copper was 3.2 mg.
Case 920. This 13-year-old Caucasian boy
complained of urinary frequency, back pain,
and fever for 3 days. There were episodes of
unexplained anemia for 1 year. Physical
examination revealed a well developed, listless child, with a sallow, waxy skin, and
erythematous papillary rash on the cheeks.
The white blood count was 6500, polymorphonuclear leukocytes 74 per cent, 6 per
cent of which were band forms, red blood
count 4,100,000, hemoglobin 12.7 Gm. The
urine was loaded with red and white blood
cells. Seven days later the white blood count
was 47,000, with 82 per cent polymorphonuclear leukocytes, of which 40 per cent were
band forms and 14 per cent metamyelocytes.
The nonprotein nitrogen was 33, icterus
index 43, CO2 combining power 12, blood
sugar 250, prothrombin 43 per cent. He died
the following day, of an acute pyelonephritis.
At autopsy, the 1420-Gm. brain revealed no
changes grossly, but microscopically there
were a few perivascular hemorrhages and an
occasional small vessel surrounded by round
cells. Just lateral and inferior to the internal
capsule a few yellow-green pigment deposits were observed, associated with pronounced looseness of structure and complete
loss of nerve cells. Dr. Courville concluded
that the acute changes in the corpus striatum
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FIG.
FIG.
FIG.
FIG.
FIGS.
2—5
2 (upper left). Case ^6). Juvenile cirrhosis. Rubeanic acid stain for copper. X 500.
3. (upper right). Case 778. Juvenile cirrhosis. Rubeanic acid stain for copper. X 250.
4. (lower left). Case 848. Juvenile cirrhosis. Rubeanic acid stain for copper. X 500.
5 (lower right). Case 915. Juvenile cirrhosis of the liver and spleen.
483
r
484
B U T T ET
Vol. 30
AL.
were caused by terminal anoxia rather than
hepatolenticular degeneration. The spleen
weighed 440 Gm. (Fig. 9). The external surface of the 1420-Gm. liver was coarsely
nodular, with many yellow areas. The extrahepatic bile ducts were patent. Sections revealed a marked increase of loosely arranged
fibrous tissue containing many capillaries
enclosing small and large nests of liver cells.
The tissue was diffusely and rather conspicuously infiltrated throughout by polynuclear
leukocytes, lymphocytes, and monocytes.
Giant cell formation was not observed. Bile
duct hyperplasia was excessive. Little iron
pigment was present in the special stained
slides; this agreed with the quantitative
estimation of 51 mg. Copper stain, however,
revealed an entirely different picture. Copper was found not only in the polygonal
cells in fine and large granules, but also in
the Kupfer cells, and in what seemed to be
histiocytes in the fibrous tissue (Fig. 10).
Liver iron was 51 mg. and copper 162 mg.
Brain iron was 32 mg. and copper 8.4 mg.
This last figure, as well as the histologic interpretation, would exclude a diagnosis of
hepatolenticular degeneration (Fig. 1).
mal appearing polygonal cells, but not in
Kupfer cells or fibrous tissue. Liver iron was
159 mg. and copper 44.5 mg. Brain iron was
37.2 mg. and copper 3.6 mg.
Case 1535. (Submitted for trace metal
studies by Dr. John T. Ellis of Cornell University School of Medicine.) This 5-year-old
boy was believed to have developed cirrhosis
of the liver and to have died of hepatic
failure. A sister died at 6 years and an uncle
at 10, both of cirrhosis of the liver.
Histologically there was a great increase
of periportal fibrous tissue, with excessive
bile duct hyperplasia and moderate infiltration of monocytes and lymphocytes
(Fig. 11). Very little iron pigment was observed, confined chiefly to the polygonal
cells. Copper stain, however, was quite striking. Much copper pigment was observed in
Kupfer cells, polygonal cells, and scattered
histiocytes, and small amounts were observed in the bile duct epithelium (Fig. 12).
Hepatic iron was 151 mg.; copper, 177.3 mg.;
and silver, 0.43 mg. The liver ash was deep
blue and required several dilutions for
measurement of copper.
Case 1058. This 9-day-old Negro boy was
flaccid, cyanotic, and had generalized edema
on admission at 4 days. The hemoglobin was
18.7 Gm., nonprotein nitrogen 87. The
Coombs test was negative. The mother was
Rh positive; her Wassermann and Kahn
tests negative. At autopsy, the 450-Gm.
brain manifested no gross abnormalities.
The liver was coarsely nodular and pale
yellow. Histologically there was a slight to
moderate increase of periportal fibrous tissue, with extension into the liver lobules,
isolating the polygonal cells into small groups.
Many large giant cells were present. In addition to blood islands, a few small collections
of lymphocytes, polynuclear leukocytes, and
eosinophils were observed. The bile canaliculi were dilated and filled with bile plugs.
Bile duct proliferation was minimal. Lymphatics and blood vessels in the periportal
spaces were conspicuously dilated. A small
amount of.iron pigment was present in the
polygonal cells and in an occasional giant
cell. Small amounts of copper pigment were
observed in giant cells and in the more nor-
HEPATIC
COPPER
LEVELS
IN
PREMATURE
INFANTS
Interest was centered on the deposition
of hepatic copper in the newborn, inasmuch
as approximately half of the patients with
juvenile cirrhosis died within the first few
days of life. Seventy-nine newborn infants
were divided into 2 weight groups, using
2500 Gm. as the point between prematurity
and full term. The average weight for the
premature group was 1.59 ± 0.07 kg., and
for the full term, 3.77 ± 0.23 kg. Hepatic
metal values for the premature infants were:
copper, 39.5 ± 2.6 mg. and zinc, 46.7 ±
2.9 mg.; copper, ranging from 112.5 to 13.0
mg. and zinc, from 105.0 to 15.0 mg. In the
term group the values were: copper, 26.49 ±
2.36 and zinc 33.16 ± 3.49 mg.; copper,
ranging from 86.0 to 6.8 mg. and zinc, from
105.0 to 15.0 mg. (Fig. 14). Where copper
was 30 mg. or more, pigment was demonstrated in the polygonal cells.
Case 601. The anatomic diagnoses of this
1-day-old Mexican girl were prematurity
and pulmonary atelectasis. Microscopically,
Dec. 1958
COPPER STORAGE DISEASES. 11
485
FIGS. 6 AND 7
FIG. 6 (upper). Case 915. Juvenile cirrhosis. Hematoxylin and eosin. X 125.
FIG. 7 (lower). Case 915. Juvenile cirrhosis. Rubeanic acid stain for copper.
X 250.
a large number of blood islands were
scattered throughout the severely congested
liver. The copper pigment was present
chiefly in the polygonal cells of the peripher-
ies of the lobules (Fig. 13). Only a small
amount of iron was present in the polygonal
cells. Hepatic iron was 140 mg. and copper
was 112.5 mg.
486
BUTT ET
Further clinical and pathologic studies
will be directed to the problem of copper
storage in the newborn. An effort will be
made to determine the serum copper levels
of mothers and infants during and after
parturition.
WILSON'S DISEASE
AL.
Vol. SO
formalin-fixed tissues and their quantities
not appreciably changed.
Case 624. This Caucasian woman, age 26,
had symptoms and sighs of Wilson's disease
for a number of years. Death was caused by
a terminal purpura. Her sister had died of
Wilson's disease. The liver manifested the
usual lobular type of cirrhosis. As has been
described previously, many of the lobules
were free of copper pigment, while the cells
of an occasional lobule contained copper
pigment in a finely granular form. Copper
pigment was not found in Kupfer cells, nor
in the fibrous tissue (Figs. 17 and 18).
Cases of 5 women and 6 men are presented.
The historic details, physical changes, and
autopsy findings in all were consistent with
a diagnosis of hepatolenticular disease. The
average age was 30, the oldest 49, and the
youngest 12 (Table 2, Figs. 1, 15, 16). Four
were from the Los Angeles County Hospital,
LAENNEC'S CIRRHOSIS
4 from other hospitals in California, and 3
Inasmuch as hepatic copper is increased
from Cornell University (provided through
the kindness of Dr. John T. Ellis, patholo- in Laennec's cirrhosis, we have included
average copper values for 56 persons. The
gist).
Brain and liver lead was elevated in the type of cirrhosis varies from the so-called
patients from Cornell, and in 1 case, a boy, subacute cirrhosis, with considerable fat and
age 12, from the White Memorial Hospital little fibrosis, to the type in which there is
in Los Angeles (submitted by Dr. Abraham much fibrosis, with or without fatty infiltraLu). In the remainder of the persons lead tion. These cases will be reported in greater
levels were within our usual limits. It is detail in a subsequent paper. The trace
interesting to note that in all organs in which metal pattern consists of a moderate increase
copper was elevated, there was an approxi- of copper and a decrease of zinc, iron, momately 10-fold increase in silver. An in- lybdenum, aluminum, cobalt, nickel, and
creased copper and silver content of liver chromium. Approximately 10 per cent of the
and kidney was observed by Rumpel in patients had increased hepatic iron, how19136' " in patients with the pseudosclerotic ever, occasionally associated with an increase
of iron in other organs, as was found in
form of Wilson's disease.
Case 1373. A white, 25-year-old man, a . hemochromatosis (Table 3).
Case 835. A'Mexican woman, age 56, had
typesetter, was admitted to the Los Angeles
County Hospital in 1938, with a diagnosis a long-standing history of alcoholism and
of encephalitis. Autopsy revealed coarsely died of liver failure. Hepatic copper was
nodular hepatic cirrhosis and bilateral 84 mg., iron 35 mg., and silver 0.08 mg.
putamen degeneration. The anatomic diag- While the case was one of a typical diffuse
nosis was hepatolenticular degeneration. fatty type of Laennec's cirrhosis, the unPortions of the liver and brain were sub- usual finding was the amount of copper
mitted to the. biochemist for heavy metal present in the liver (Fig. 19). The brain
studies, including copper and lead. It was copper was 6.2 mg., iron 16.8 mg., and silver
reported that the lead content was increased 0.04 mg.
in the liver, kidneys, and brain, and that
CONTROL MATERIAL
copper was not present. Fortunately, blocks
of the liver and brain were found in our wet (Including all pathologic states except
copper and iron storage diseases)
tissue file. Spectrographic analysis revealed
that the lead content was within normal
These 96 cases consist largely of heart dislimits and the copper increased in the brain ease, carcinomas of various sites, chronic
and liver.16 This experience indicated that degenerative diseases, and acute infections
trace metals are rather firmly bound in (Table 4).
Dec. 1958
48
COPPER STORAGE DISEASES. II
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F I G S . 8 AND 9
F I G . 8 (upper). Case 915. Juvenile cirrhosis. Rubeanic acid stain for copper.
X 250.
F I G . 9 (lower). Case 920. Juvenile cirrhosis of the liver and kidney.
*
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jtfj^-f
FIGS.
FIG.
FIG.
FIG.
FIG.
X 250.
10
11
12
13
-/*^
S » V d-
• ?
10—13
(upper left). Case 920. Juvenile cirrhosis. Rubeanic acid stain for copper. X 500.
(upper right). Case 1535. Juvenile cirrhosis. Hematoxylin and eosin. X 75.
(lower left). Case 1535. Juvenile cirrhosis. Rubeanic acid stain for copper. X 500.
(lower right). Case 601. Hepatic copper in premature infant. Rubeanic acid stain for copper
4SS
Dec. 1958
4S9
COPPER STORAGE D I S E A S E S . II
A.
BIRTH TO 3 MONTHS
PREMATURE INFANTS
B.
AV WT. = 1.59 Ko. * 0.072.
BIRTH TO 3 MONTHS
MATURE INFANTS
AV. WT. = 3.77 Kq. ±0.228
C.
3 MONTHS TO 1YEAR.
D.
1 TO 100
YEARS
AV. = AVERAGE
D-NUMBER. OF CASES
| AV. 16.7 t 2.68
A- t
| AV. 39.3
A/. 33.16 15.49
| atjiMa t u t ]
[»<1
B.
Zn
Cu
L
Cu IT>"
1*1371
-
D.
± 2.60
Zn ^_
r„
0
1=1
B
| W. 27.6 t i . < «
1
|
| «/. -4.53 to. 589
10
20
1
JO
40
50
60
70
80
90
100
110
120
Mo,, PER. 100 oms. DRY TISSUE
FIG. 14. Storage of copper and zinc in the liver
DISCUSSION
Routine examination of human organs for
selected trace elements has revealed that
copper is moderately increased in the liver
in a number of disease conditions without
significant alteration of other metal levels,
except for iron. Yet, from a storage standpoint, copper seems less related to iron than
does calcium, lead, molybdenum, or aluminum. For instance, hepatic copper is usually
increased in Laennec's cirrhosis, whereas
iron levels are usually decreased. Moreover,
if hepatic iron levels are elevated in
Laennec's cirrhosis, elevated copper levels
remain unaltered, but lead, molybdenum,
and calcium are increased. Qualitatively,
then, the pattern is one of hemochromatosis
and probably represents an incipient stage
of the disease.
These small increments of hepatic copper
occur in disease states reported to have increased levels of plasma copper, such as
chronic infections, leukemic states, anemias,
and Hodgkin's disease.8 Inasmuch as these
diseases are not linked with an irregular protein and amino acid metabolism, or accompanied by excess copper storage, we prefer
to group them as diseases with secondary
hepatic copper storage. Probably such copper increases represent mobilization of
copper from involved tissues. Wilkinson,17
speculating about increased copper plasma
in these diseases, stated, " . . . the copper is
related to the antigen-antibody system and
functions in some way in the defense mechanism of the body . . . the altered copper metabolism is a manifestation of the increased
metabolic activity of the cells or . . . copper
is simply a product of destruction of the
cells."
Little attention has been directed, as yet,
to the study of copper levels in cases of
juvenile cirrhosis. It was within this group
that we observed hepatic levels much greater
than in Wilson's disease. Less hepatic copper
was observed in cirrhosis of infants less than
1 year than in older children with cirrhosis.
No attempt was made to classify juvenile
cirrhosis, yet there seemed to be 3 types in
our material: (1) nodular cirrhosis with excessive bile duct hyperplasia, (2) nodular
cirrhosis with little or nearly absent bile
duct hyperplasia, and (3) giant cell disease
of the liver.
Histochemical studies of the livers were
of considerable interest. Instead of the more
uniform distribution of copper present only
in the polygonal cells of an occasional lobule,
as seen in Wilson's disease, the distribution
of copper pigment was quite irregular, but
diffuse. Not only were the polygonal cells
involved, but copper also was found in the
0.411
32.9
0.84
46.3
13.2
2.36
0.13
100.0
11.6
0.84
0.12
2.90
0.50
345.0
27.8
0.78
0.20
85.5
17.2
Range
8
8
3
8
8
8
8
8
78.0
32.4
0.42
0.04
52.5
20.2
32.2
3.84
0.21
Range
6.9
0.72
0.86
0.50
70.0
36.8
10.9
0.118
0.17
0.06
13.6
35.5
0.26
55.5
1.38
Number Arithmetic
of cases average
Brain
3
3
2
3
3
3
3
3
0.02
32.3
10.73
3.66
0.22
57.6
0.22
Number Arithmetic
of cases average
Heart
0.020
14.3
6.6
0.12
ND*
39.0
21.1
0.032
0.168
0.230
77.5
46.0
0.26
0.13
4.7
2.4
Range
5
5
2
5
5
5
5
5
0.16
52,2
0.16
13.7
21.5
0.25
85.3
0.86
Number Arithmetic
of cases average
Kidney
(Values i n d i c a t e d are mg. per 100 Gm. of dry
* N D = n o t d e t e c t e d within t h e limits of the m e t h o d .
Ag
Ca
Mo
24.5
Zn
0.47
Mn
45.4
108.6
Fe
Cu
1.35
Number Arithmetic
of cases average
Liver
Pb
Element
TABLE 2 .
91.0
11.5
0.46
0.06
17.5
6.9
0.20
0.12
27.7
4.4
1.23
0.28
148.0
50.0
0.46
0.14
Range
tissue)
TRACE METAL PATTERN IN WILSON'S DISEASE
5
5
3
5
5
5
5
5
0.05
49.9
0.52
13.26
17.56
0.29
278.0
1.58
Number Arithmetic
of cases average
Lung
4.42
0.15
470.0
125.0
0.52
0.16
39.0
0.9
19.2
9.6
0.64
0.39
92.0
14.8
0.08
0.02
Range
5
5
2
5
5
5
5
5
0.04
37.4
0.16
16.2
3.14
0.29
223.7.
0.75
Number Arithmetic
of cases Average
Spleen
1.56
0.22
498.0
118.0
0.48
0.16
• 6.1
1.4'
34.5
4.6
0.20
0.12
46.5
18.5
0.074
0.021
Range
Dec. 1958
A.
B.
C
D.
A. I"
B-
491
COPPER STORAGE DISEASES. 11
c
IAV.OJSS
s
iff
E3
^
I Ay CMII
U AV. 10.15 t l . B 9
UTS.061 1 0 . 0 1
C.
D.
JUVENILE CIRRHOSIS
HEPATOLENTICULAR DISEASE
(WILSON)
LAENNEC CIRRHOSIS
CONTROL-AVERAGE ALL DISEASES
| AV. 4 . S 5
Cu
s
AV. = AVERAGE
D - N U M B E R . OF CASES
1 aS89
Aq 7 AV. aos* taoos
102O30405060708O9010O:102030'10
Cu
$060708090200
01 0.2 0.J 0.4 05 06 07 08 09 W 11 IX 1.5 14 1.5 16 17 18 19 2.0
Mq. PER. 100 qms. DRY TISSUE
F I G . 15. Storage of silver and copper in t h e liver
I . JUVENILE CIRRHOSIS
H. HEPATOLENTICULAR DISEASE (WILSON)
M. AVERAGE METAL DEPOSITION - ALL DISEASES
*•
I , . ;.
LIVER
1
S O
BRAIN
..-.-fe*3**.aS^1
®
nL3/una 10.209
i | • l * 7 . ? S '•"••I
l«J
/JU2A1
I
KIDNEY
I
IV. 4.53
10.589
Q]<US.T3
H
S
• • l - . g
(S)
El
If,
3T„ I - '
-•:•-•
tais
AV 1 . 9 4
a
l£j
I | -•SMS&
LUNG
HEART
SPLEEN
T
-
•
•
>
'
•
'
&
nL *11.4«
*
iscSil
10.091
I Emio
U J>m 3.6t
E)
(Tl
J i g * 2.58
to.106
nJI3wj.io
IE)
r.
A/. - A/ERA&&
* NUMBER OF CASES
•
GU
s
[Ml
10 20 30 40 5 0 6 0 7 0 8 0 9 0 1 0 0 1 0 2 0 5 0 4 0
a | A V . 1.04.
0
El
t 0.09
50607080
90200
Mq. PER. 100 qms. DRY TISSUE
F I G . 16. Levels of copper in various organs
Kupfer cells, histiocytes, and in a few
instances in the epithelial cells of the bile
ducts. The pigment was coarsely granular
and in greater abundance around the peripheries of the lobules. In some instances,
the polygonal cells contained iron as well as
copper pigment. Both pigments were also
found in giant cells.
In juvenile cirrhosis, with the exception of
case 1535, the brain levels for copper and
silver were within normal limits. No brain
tissue was received for case 1535; however,
the copper distribution in the liver was
similar to the other cases of juvenile cirrhosis. I t would serve no purpose at present to
classify these cases as variants of Wilson's
disease without complete biochemical
studies during life and at autopsy. Obviously, the problem of juvenile cirrhosis needs
considerable clarification clinically, patho-
492
BUTT ET
AL.
Vol. 30
FIGS. 17 AND 18
FIG. 17 (upper). Case 624. Wilson's disease. Rubeanic acid stain for copper. X 75.
FIG. 18 (lower). Case 624. Wilson's disease. Rubeanic acid stain for copper. X 125.
logically, and chemically before the pathogenesis will become evident and etiologic
factors demonstrable.
There are a number of references in the
literature regarding the levels of copper in
Wilson's disease.3"5'7 Our average values for
liver and brain are in agreement. Elevated
copper levels in this disease are observed in
Heart
56
56
56
56
56
35
30
15
0.06
0.16
21.02
0.59
10.14
13.87
0.55
65.16
0.07
10.91
0.04
1.89
1.12
0.03
1.95
0.01
26
14
50
50
50
50
50
29.05
0.23
3.38
5.70
ND*
42.3
0.03
0.14
0.0005
6.41
0.18
3.38
0.02
0.26
0.53
27
10
52
52
52
52
52
2.18
9.37
. ND
33.09
0.03
0.14
30.46
0.23
Number ArithNumber Arithmetic Standard Number Arithmetic
metic Standard
of cases average
of cases average
error
error
of cases average
Brain
* N D = not d e t e c t e d within t h e limits of the m e t h o d .
Ag
Fe
Mn
Cu
Zn
Mo
Ca
Pb
Element
Liver
TABLE 3
Lung
Spleen
5.03
0.03
2.90
0.03
0.16
0.66
56
55
56
54
56
21
27
12
0.02
33.58
0.52
1.77
19.14
0.14
125.07
0.34
15.28
0.03
3.21
0-.04
0.12
1.31
26
9
51
51
51
51
51
0.30
120.07
0.21
1.36
6.95
ND
55.68
0.02
5.49
0.03
14.18
0.04
0.15
0.50
25
9
50
50
50
50
49
0.48
144.51
0.15
1.47
7.89
ND
27.07
0.02
2.38
0.09
21.72
0.01
0.22
0.64
ArithStandard Number metic Standard Number Arithmetic Standard Number Arithmetic Standard
of cases average
of cases average
error
error
error
error
of cases average
Kidney
TRACE METAL PATTERN IN LAENNEC'S CIRRHOSIS
(Values i n d i c a t e d are mg. per 100 Gm. of dry tissue)
494
BUTT ET
~~*r
rrr—~~~
J 'r<*
V. <rt
Vol. SO
AL.
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-y
J . •*»'., '_Y,'
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** 4.™*"**
-'%
' .**»<*-
-Vt£;
\ii£>^di
F I G . 19. Case SS5. Laennec's cirrhosis. Rubeanic acid stain for copper. X 500.
the liver, brain, kidneys, and to a lesser extent, in the lungs. Relatively normal values
for copper are observed in the spleen and
heart. Of unusual interest, however, is the
elevation of silver in the same organs in
which there is an increase in copper. Moreover, silver levels of the heart and spleen are
within normal limits. A distinct histologic
alteration of the tubular epithelium is observed in Wilson's disease, the details of
which will be published later.
While the average hepatic copper level in
Laennec's cirrhosis is much lower than in
juvenile cirrhosis or Wilson's disease, in an
occasional patient the liver contained levels
of copper equal to or above the average
value for Wilson's disease.
A common factor in juvenile cirrhosis,
Wilson's disease, and Laennec's cirrhosis is
the capability to store copper. Manifestly,
this is not of etiologic significance. It is possible that there exists an equivalent genetic
irregularity or developmental alteration of
protein or enzyme arrangements that result
in an excess storage of copper and silver
when modified by an inciting agent. As has
been indicated, excess retention of copper
was observed, also, in some premature as
well as full term infants.
The search for irregular copper-binding
proteins may prove fruitful in these diseases,
and in the newborn. Uzman and his associates14 have demonstrated, in Wilson's disease, livers with an abnormal protein fraction with increased affinity for copper. Porter
and Folch10 also have observed an abnormal
copper-protein combination in the brains of
persons with hepatolenticular degeneration.
No record is found of liver-protein studies of
juvenile cirrhosis, Laennec's cirrhosis, or in
premature infants with excessive storage of
hepatic copper.
High hepatic levels of copper and iron are
reported in the normal healthy newborn. 2,17
Contrary to Underwood,12 who states, "No
evidence of any appreciable fetal stoi'age of
zinc occurs with iron and copper," we find
that zinc also is elevated in the newborn. He
writes further, "In man and the rat, rabbit,
guinea pig, dog and cow, maximum concentrations (of copper) occur at or shortly after
birth, but in the pig and chick the peak occurs somewhat earlier in embryonic life."
Analysis of our data reveals that higher
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496
B U T T ET
hepatic levels of copper and zinc are present
in premature than in term infants. In some
instances, the storage level of copper equals
the average copper level of juvenile cirrhosis.
The question arises: Does the mother have
excess mobilization of copper owing to pregnancy, or pregnancy and infection, or are
the fetal liver proteins altered by maternal
or genetic irregularities resulting in a protein
which abnormally chelates copper? If the
mother ingests or mobilizes unusual amounts
of copper, the fetal storage should be higher.
Dietary factors, such as variations in the
quantity of molybdenum and inorganic sulfate, may be responsible for irregularities of
storage of copper.12
There is a dearth of information regarding
copper metabolism in the fetal stage of man.
CONCLUSIONS
1. Spectrographic analyses of liver, kidneys, and brain tissue reveal a 10-fold increase in silver, with copper storage, in
Wilson's disease.
2. Of the organs studied, excess copper
and silver storage was observed only in the
liver, brain, and kidneys; the spleen and
heart had normal levels.
3. A geographic variation of lead storage
was observed in Wilson's disease.
4. AVhereas in juvenile cirrhosis the
hepatic metal pattern had points of similarity to Wilson's disease, the brain pattern
was within normal limits.
5. The distribution of copper pigment in
juvenile cirrhosis was unlike the distribution
in Wilson's disease.
6. The average hepatic values of copper
for premature infants under 2500 Gm. was
greater than for infants over 2500 Gm.
7. Excess hepatic storage of copper was
observed in an occasional premature and full
term infant.
SUMMAIUO INT I N T E R L I N G U A
1. Analyses spectrographic de histos
hepatic, renal, e cerebral revel a un decuple
augmento de argento, con le immagasinage
de cupro, in morbo de Wilson.
2. Inter le organos studiate, excessos de
immagasinage de cupro e argento esseva
Vol. 30
AL.
observate solmente in le hepate, le cerebro,
e le ren. In splen e corde, le nivellos esseva
normal.
3. Un variation geographic del immagasinage de plumbo esseva observate in morbo
de Wilson.
4. Quanto al presentia de metallos, cirrhosis juvenil e morbo de Wilson ha similaritates in le situation hepatic seel non in le
situation cerebral. Iii cirrhosis juvenil le
representation del metallos in le cerebro se
monstrava normal.
5. Le distribution de pigmento a cupro in
cirrhosis juvenil differeva ab illo in morbo de
Wilson.
6. Le valores medie de cupro hepatic in
infantes immatur de pesos de minus que
2500 g esseva plus grande que le valores
correspondente in infantes de pesos de plus
que 2500 g.
7. Excessos del immagasinage hepatic de
cupro esseva observate sporadicamente in
infantes prematur e in infantes nascite a
termino.
REFERENCES
1. B U T T , E . M . , N U S B A U M , R . E . , G I L M O U R , T .
C , AND D I D I O , S. L . : Use of t h e emission
spectrograph for study of inorganic elements
in human tissues. Am. J . Clin. P a t h . , 24:
385-394, 1954.
2. B U T T , E . M . , N U S B A U M , R. E . , GILMOUR, T.
C , AND D I D I O , S. L . : Trace metal patterns
in disease states. I . Hemochromatosis and
refractory anemia. Am. J . Clin. P a t h . , 26:
225-242, 1956.
3. CAB.TWR.IGHT, G. E . , H O D G E S , R. E . , G U B L B R ,
C. J., M A H O N E Y , J . P . , D A U M , K . , W I N TROBE, M . M . , AND B E A N , W. B . : Copper
metabolism in Wilson's disease. J . Clin.
Invest., 33: 1487-1501, 1954.
4. CUMINGS, J . N . : T h e copper and iron content
of the brain and liver in normal and in
hepatolenticular degeneration. Brain, 7 1 :
410-415, 194S.
5. CUMINGS, J . N . : Copper storage in hepatolenticular degeneration and allied diseases.
Proc. Royal Soc. Med., 47: 152-154, 1954.
6. D E N N Y - B R O W N , D . ,
AND P O R T E R ,
H.:
The
effect of BAL (2,3-dimercaptopropanol) on
hepatolenticular degeneration (Wilson's disease). New England J . Med., 245: 917-925,
1951.
7. G R E E N , C. L.: Histochemical demonstration of
copper in a case of hepatolenticular degeneration. Am. J . P a t h . , 3 1 : 545-553, 1955.
S. M C E L R O Y , W. D . , AND G L A S S , B .
(Editors):
Copper Metabolism—A Symposium on Animal, P l a n t and Soil Relationships. Baltimore: Johns Hopkins Press, 1950, pp. 451.
Dec. 1958
C O P P E R STORAGE
9. N U S B A U M , R. E . , B U T T , E . M., G I L M O U R , T .
C , AND D I D I O , S. L . : Analysis of biological
materials with a quantometer. (Paper presented a t meeting of Am. Chemical Soc.
April 1958).
10. P O R T E R , H . , AND FOLCH, J . : Brain
copper-
protein fractions in t h e normal and in Wilson's disease. A. M . A. Arch. Neurol. &
Psychiat., 77: 8-16, 1957.
11. R U M P E L , A.: Ueber das Wesen unci die Bedeutung der Leberveranderungen und der
Pigmentierungenbei den damit verbundenen
Fallen von Pseudosklerose zugleich ein
Beit rag zur Lehre von der Pseudosklerose
(Westphal-Striimpell). Deutsche
Ztschr.
Nervenh., 49: 54-73, 1913.
12. UNDERWOOD, E . J . : Trace Elements in H u m a n
and Animal Nutrition. New York: Academic Press, I n c . , 1956, 429 pp.
13. UZMAN, L. L . : I n t r a h e p a t i c distribution of
DISEASES. II
497
copper in relation to t h e pathogenesis of
hepatolenticular degeneration. A. M . A.
Arch. P a t h . , 64: 464-479, 1957.
14. UZMAN, L. L., IBER, F. L., AND CHALMERS,
T. C . : Mechanism of copper deposition in
liver in hepatolenticular
degeneration
(Wilson's disease). Am. J . M . S c , 231: 51151S, 1956.
15. V A L L B B , B . L., W A C K E R , W. flH^., BARTHOLO-
MAY, A. F . , AND HOGH, F . E ^ Z i n c metabolism in hepatic disfunction. New England
J. Med., 257: 1055-1065, 1957.
16. V O N H A G E N , K . 0 . , AND B U T T , E . M . : H e p a -
tolenticular degeneration in a case with
disturbed protein metabolism and probable
lead intoxication. Bull. Los Angeles Neurol. S o c , 5: 107-112, 1940.
17. WILKINSON, J . F . : Modern Trends in Blood
Diseases. New Y o r k : Paul B. Hoeber, I n c . ,
1955, 359 pp.

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