Clinical, morphologic, and chemical studies on copper toxicosis of Bedlington Terriers


Twedt, D.C.; Sternlieb, I.; Gilbertson, S.R.

Journal of the American Veterinary Medical Association 175(3): 269-275

1979


In a study of 90 Bedlington Terriers, 68 had a defect that resulted in the accumulation of toxic excesses of copper in the liver. Concentrations of copper were 5 to 50 times that of clinically normal mongrel dogs. The bulk of this excess copper was sequestered in lysosomes. When copper concentrations exceeded 2000 mu g/g dry liver, progressive signs of functional and morphological disturbance appeared as focal hepatitis, chronic active hepatitis, and ultimately cirrhosis. The disorder, which appears to be inherited, could only be diagnosed by liver biopsy. It was latent for many years in some dogs, but led early in life to acute or chronic hepatic disease and death in others.

O
I
meal,
Morphologic,
and
Chemical
Studies
on
Copper
Toxicosis
Bedlington
Terriers
id
C.
Twedt,
DVM;
Irmin
Sternlieb,
MD;
Steven
R.
Gilbertson,
DVM
SUMMARY
In
a
study
of
90
Bedlington
Terriers,
68
had
a
defect
that
resulted
in
the
accumulation
of
toxic
excesses
of
copper
in
the
li
ver.
Concentrations
of
copper
were
5
to
50
times
that
of
clinically
normal
mongrel
dogs.
The
bulk
of
this
excess
copper
was
sequestered
in
lysosomes.
When
copper
concentrations
exceeded
2,000
p.g/g
dry
liver,
progressive
signs
of
functional
and
morphologic
disturbance
appeared
as
focal
hepatitis,
chronic
active
hepatitis,
and
ultimately
cirrhosis.
The
disorder,
which
1
appears
to
be
inherited,
could
only
be
diagnosed
by
liver
oiopsy.
It
was
latent
for
many
years
in
some
dogs
but
led
early
in
life
to
acute
or
chronic
hepatic
disease
and
death
in
others.
THE
NATURE
of
the
progressive
and
fatal
liver
disease
known
to
affect
some
Bedlington
Terriers
1
was
dis-
covered
when
histochemical
staining
revealed
an
abun-
dance
of
copper
in
dark
granules
in
hepatocytes.
2
In
this
report,
we
describe
clinical,
pathologic,
and
From
the
Division
of
Genetic
Medicine
and
the
Liver
Research
Center,
Albert
Einstein
College
of
Medicine,
1300
Morris
Park
Ave,
Bronx,
NY
10461
rwedt,
Sternlieb),
and
The
Animal
Medical
Center,
510
E
62nd
St,
New
lerit,
NY
10021
(Twedt,
Gilbertson).
Dr.
Twedt's
address
is
Department
of
Clinical
Sciences,
College
of
Veterinary
Medicine
and
Biomedical
Sciences,
Colorado
State
University,
Fort
Collins,
CO
80523.
Dr.
Gilbertson's
present
address
is
Memorial
Sloan-Kettering
Cancer
Center,
New
York,
NY
10021.
Supported
by
grants
from
the
National
Institutes
of
Arthritis,
Metabolism
an
d
Digestive
Diseases
(AM
1059
and
AM
17702),
the
National
Cancer
In-
titute
ICA
6576),
and
the
Foundation
for
the
Study
of
Wilson's
Disease,
Inc.
The
authors
thank
the
late
Maryanna
Padula
for
initiating
this
work
be-
.
.suse
of
her
profound
interest
in
the
welfare
of
Bedlington
Terriers;
Janet
lever.
RN,
Deanne
Segale,
Barbara
H.
Whiting,
and
Constance
Willemsen
r
cooperation;
Phyllis
Grushoff
and
Edie
Korotkin
for
technical
assistance;
Gerald
F.
Johnson,
The
Animal
Medical
Center,
New
York,
NY,
and
W.
Lybrook,
HiWay
Veterinary
Hospital,
Scottsdale,
Ariz,
for
obtaining
of
the
liver
biopsy
specimens;
Dr.
Sidney
Goldfischer,
Albert
Einstein
College
of
Medicine,
Bronx,
NY,
for
performing
the
histochemical
studies;
and
Nelson
Quintana,
Albert
Einstein
College
of
Medicine,
Bronx,
NY,
for
ob-
oimng
the
electron
micrograph.
of
the
american
veterinary
medical
association
chemical
studies
by
means
of
which
we
could
assign
each
of
90
Bedlington
Terriers
to
one
of
two
classes:
(1)
unaffected
dogs,
with
normal
appearing
hepatocytes
on
light
microscopic
examination
and
hepatic
copper
concentrations
less
than
360
pg/g
dry
tissue;
and
(2)
af-
fected
dogs,
with
prominent
dark
granules
in
the
hepato-
cytes
and
much
higher
hepatic
copper
concentrations,
ranging
from
850
to
10,600
pg/g
dry
tissue.
Materials
and
Methods
The
study
involved
44
male
and
46
female
Bedlington
Terriers
ranging
in
age
from
6
months
to
15
years.
A
clin-
ical
history
and
physical
examination
were
obtained
for
each
dog.
The
corneas
of
10
affected
dogs
were
examined
for
copper
deposits,
using
a
hand-held
slit
-lamp
biomicro-
scope.
Routine
serum
biochemical
analyses,'
including
de-
terminations
of
alanine
aminotransferase
(serum
glutamic-
pyruvic
transaminase
(sem')
)
,
were
performed
in
75
dogs
by
the
clinical
laboratory
of
The
Animal
Medical
Center.
Results
obtained
elsewhere
were
not
included
in
our
anal-
ysis.
Under
peritoneoscopic
control,
liver
biopsy
samples
were
obtained
in
58
dogs
with
copper
-free
needles°
and
in
20
by
wedge
resection
during
laparotomy.
There
were
12
necropsy
specimens.
Each
sample
was
divided
into
two
portions,
one
of
which
was
used
for
chemical
analysis
and
the
other
for
morphologic
studies.
Normal
Control
Dogs
—Hepatic
copper
concentrations
were
determined
in
15
clinically
and
biochemically
normal
mixed
-breed
dogs
of
random
size,
age,
and
sex
with
histo-
logically
proved
normal
livers.
Chemical
Analysis
—Quantitative
copper
determination
was
performed
on
specimens
dried
at
65
C
in
a
vacuum
oven
for
12
hours
followed
by
wet
digestion
and
spectro-
photometric
analysis'
.Serum
copper
values
were
deter-
mined
by
atomic
absorption
spectrophotometry.
*
SMA
12/60
Autoanalyzer,
Technicon
Instruments
Corp,
Tarrytown,
NY.
b
Tru-
Cut
biopsy
needles,
Travenol
Laboratories
Inc,
Deerfield,
Ill.
gust
1,
1979
269
Light
Microscopy
—All
formalin-fixed
li
ver
specimens
were
sectioned
at
6µm
and
stained
with
hematoxylin
and
eosin.
Twenty
sections
were
stained
with
rubeanic
acid
or
rhodanine
for
copper.
Other
selected
sections
were
stained
with
periodic
acid
-Schiff
for
glycoproteins,
Masson's
tri-
chrome
for
collagen,
Fontana's
stain
for
melanin,
orcein
for
"copper
protein,'"
and
Perls'
stain
for
iron.
Paraffin
-
embedded,
unstained
sections
were
examined
for
autoflu-
orescence.
The
abundance
and
prominence
of
lysosomal
granules
in
sections
obtained
from
affected
dogs
was
graded
by
one
of
us
(SRG)
without
knowledge
of
either
clinical
or
chemical
results
pertaining
to
the
specimens
under
study.
Electron
Microscopy
—Biopsy
specimens
were
sectioned
and
processed
according
to
previously
published
methods.'
Fine
sections
obtained
from
two
affected
dogs
were
placed
on
titanium
grids
and
studied
in
an
electron
microscope
equipped
with
an
electron
dispersion
spectrophotometer.
Results
The
histologic
fi
ndings
in
the
liver
samples
and
the
hepatic
copper
concentrations
of
22
Bedlington
Terriers
ranging
in
age
from
6
months
to
15
years
were
indis-
tinguishable
from
those
of
the
normal
mixed
-breed
dogs.
From
91
to
358
/
kg
of
copper/g
dry
liver
(mean
±
SD,
206
±
56
µg/g)
was
found
in
the
Bedlington
Terriers,
and
from
94
to
270
p.g
of
copper/g
dry
tissue
(mean
±
SD,
190
±
56
peg)
was
found
in
the
15
mixed
-breed
dogs.
In
the
other
68
Bedlington
Terriers,
the
hepatic
copper
concentrations
ranged
from
850
to
10,600
p.g/g
dry
weight,
with
the
lower
concentrations
found
in
the
youngest
dogs,
at
about
6
months
of
age
increasing
to
a
peak
mean
value
at
6
years
of
age
(Fig
1).
The
mean
hepatic
copper
concentration
for
dogs
more
than
10
years
old
was
significantly
lower
(Student's
t
test,
d..
Fig
2
—Photomicrograph
of
an
orcein-stained
section
of
a
liver
biopsy
specimen
from
a
7
-
month
-old
Bedlington
Terrier
bitch.
Coarse
cytoplasmic
granules
(arrowheads)
are
in
the
hepatocytes
(graded
1+
on
the
number
of
granules
seen
on
the
biopsy).
Hepatic
cop-
per
concentration
was
1,038
;Lag
dry
tissue.
X
480.
Inset
—Acid
phosphatase
activity
in
hepatocellular
granules
similar
in
location
to
those
staining
with
orcein.
X
400.
4
E
O
0
0
U
❑9
<1
1-2
2-4
4-6
6-8
8-10
>10
AGE
GROUPS
(YEARS)
Fig
1
—Hepatic
copper
concentration
and
numbers
of
dogs
(in
black
circles)
in
different
age
groups
of
affected
Bedlington
Terriers.
The
group
of
dogs
less
than
2
years
old
was
divided
into
two
subgroups
of
6-
to
12
-month
-old
and
1-
to
2
-year
-old
dogs
in
order
to
empha-
size
the
rapid
increase
in
the
hepatic
copper
concentration
taking
I
place
during
maturation.
The
height
of
the
bar
represents
the
mean
/
SEM)
for
each
age
group.
270
JAVMA,
Vol
175,
No
3
fi
0.01)
than
that
of
the
4-
to
6
-year
-old
group.
In
0
specimens,
with
or
without
signs
of
liver
injury,
pr
ominent
dark
granules
resembling
lipofuscin
were
dif-
k
ise
ly
distributed
in
the
cytoplasm
of
hepatocytes
(Fig
2).
These
granules
gave
positive
reactions
for
acid
phos-
Pha
t
a
se
in
Gomori's
medium,
indicating
that
they
were
iysosomes
(
Fig
2
inset
)
,
and
gave
positive
results
for
cop
p
e
r
with
rubeanic
acid
or
rhodanine
in
each
of
a
limited
number
of
slides
tested
with
these
stains.
They
also
contained
periodic
acid
-Schiff
-positive
material,
in-
•dicative
of
glycoproteins;
melanin,
as
known
by
Fon-
I
tana's
stain;
copper
-binding
protein,
as
visualized
with
)
Shikata's
orcein;
but
no
iron
when
Perls'
reaction
was
used.
The
granules
fluoresced
at
570
nm
when
excited
by
ultraviolet
light.
Under
the
electron
microscope
they
appeared
as
large,
irregular,
electron
-dense
bodies.
Lim
-
1
icing
membrane
could
not
be
resolved
because
of
the
-,
I
density
of
the
lysosomal
matrix.
Electron
dispersion
spectrophotometry
confirmed
the
presence
of
copper
in
these
granules
(Fig
3).
Hepatic
biopsy
specimens
from
the
68
Bedlington
i
Terriers
with
elevated
copper
concentrations
were
classi-
1
fl
ed
into
four
groups
according
to
the
histopathologic
severity
of
liver
injury.
Group
I
comprised
11
specimens
without
sign
of
li
ver
injury.
Some
of
the
sections
obtained
from
younger
dogs
contained
fewer
granules
and
these
were
predom-
inantly
centrilobular.
Group
II
included
25
biopsy
specimens
generally
.
characterized
by
focal
hepatitis:
scattered
microfoci
of
acute
or
subacute
necrosis
of
hepatocytes,
displaying
karyolysis
rather
than
pyknosis,
and
randomly
distrib-
uted
in
the
hepatic
lobules.
Only
in
some
of
these
specimens
were
these
foci
accompanied
by
Kupffer
cell
hyperplasia,
infiltrates
of
neutrophils
and
macrophages,
or
occasionally
with
early
fi
brosis
extending
from
portal
areas.
The
cytoplasmic
granules
were
more
numerous
than
in
group
I
and
were
diffusely
distributed
through-
out
the
lobule
(Fig
4).
Fig
4
—Photomicrograph
of
a
needle
bi-
°
b
Ps
Y
specimen
of
liver
from
a
2
-year
-old
Bedlington
Terrier.
By
unfocusing
the
condenser
of
the
microscope,
the
stip-
15li
ng
of
the
h
e
p
a
t
oce
ll
u
l
ar
cy
t
o
pl
asm
d
ue
,
Pigment
granules
is
enhanced.
Notice
focal
accumulations
of
inflammatory
cells,
11,'.14•
CatiVe
of
focal
hepatitis.
Hepatic
cop-
ier
concentration
was
5,438
/Lag
dry
'issue.
H&E
stain;
X
250.
08
HS
S~EV.CH
5
X.
18
Fig
3
—Electron
micrograph
showing
portion
of
a
hepatocyte
from
an
affected
Bedlington
Terrier
(L,
lysosomal
granule;
M,
mitochondrion;
P,
peroxisome
with
crystalline
core).
Notice
the
heterogeneous
con-
tents
of
the
membrane
-bounded,
electron
dense
body
(L).
Inset
The
x-ray
emission
spectrum
of
a
450
nm
spot
obtained
from
a
sim-
ilar
body
from
an
unstained
fine
section.
Energy
levels
in
keV
are
shown
in
abscissa.
This
spectrum
shows
a
characteristic
copper
peak
(arrow),
which
exceeds
that
recorded
from
adjacent
cytoplasm.
The
peak
on
the
right
is
that
of
the
overlapping
Cu
-Os
spectra.
The
titanium
peak
on
the
left
originates
from
the
grid.
Osmium
tetroxide;
Epon;
Philips
EM
301
with
EDAX
(courtesy
of
Irene
Piscopo,
Philips
Electronic
Instruments,
Mount
Vernon,
NY).
x
26,000.
fo
ti
111
0
0
4
ANA
A
ugust
I,
1979
271
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17r-
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$
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ctip
'l
e:
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4
,
41
.
to
10
a
Fig
5
—Photomicrograph
of
necropsy
specimen
from
a
9
-year
-old
Bedlington
Terrier
bitch,
demonstrating
formation
of
nodules,
marked
infiltration
of
inflarn•
matory
cells,
early
fibrosis,
and
an
abun•
dance
of
pigment
granules.
Hepatic
cop=
per
concentration
was
10,562
Ag/g
dry
tissue.
H&E
stain;
X
250.
4*
.104
3
,iiir
t
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Fig
6
—Photomicrograph
of
a
necropsy
specimen
of
liver
from
a
9
-year
-old
Bedlington
Terrier.
Regenerative
nodules
and
extensive
fibrosis
have
replaced
the
normal
lobular
architecture.
There
is
a
moderate
inflammatory
cell
infiltrate.
The
lymphatics
and
blood
vessels
are
'
11-
lated.
The
fatty
changes
are
probably
preterminal.
Hepatic
copper
concentration
was
6,898
µgig
dry
tissue.
H&E
stain;
x
110.
Inse
t
Same
section
viewed
with
unfocused
condenser,
demonstrating
the
abundance
of
pigment
granules
(grade
4+;
arrows).
H&E
stain;
X
25
'
Group
III
included
21
biopsy
specimens
with
fea-
tures
of
chronic
active
hepatitis:
focal
and
piecemeal
necrosis
with
infiltrates
of
lymphocytes,
plasma
cells,
and
polymorphonuclear
cells
extending
mostly
from
th
e
portal
areas.
Degenerative
changes
of
hepatocytes
--
;
swelling,
clumping
of
the
cytoplasm,
or
the
presence
oa
272
JAVMA,
Vol
175,
No.
4
'1
3
fig
7
—Liver
from
a
10
1/2
-year
-old
Bedlington
Terrier
with
the
coarsely
hobnailed
appearance
of
micro-
and
macronodular
cirrhosis.
12
11
10-
9-
8-
7
6—
4*
5
.
4—
mg
/g
dry
liver
3
2
1
•••
I
I
1+
2+
3+
4+
F
'9
8
—Relationship
of
light
microscopic
grading
of
cytoplasmic
gran-
ules
(based
on
number
and
prominence)
and
quantitative
hepatic
Co
pper
determinations
in
68
affected
Bedlington
Terriers.
The
hor-
izontal
bars
represent
the
mean
copper
concentration
for
each
grade.
li
Pid
droplets
—were
often
noticed.
Bridging
necrosis
'vas
occasionally
seen.
Adjacent
to
the
areas
of
necrosis
Were
foci
of
intracellular
and
intracanalicular
bile
stasis.
The
Kupffer
cells
were
prominent
and
contained
nu-
merous
pigmented
inclusion
bodies.
Fibrosis,
which
var-
ied
considerably
in
amount
from
specimen
to
specimen,
formed
portal-to-portal
area
bridges
in
some
and
ex-
tended
from
the
portal
areas
into
the
lobules
in
others
Fig
5).
Numerous
dark
granules
were
present
in
most
I
riePatocytes.
Group
IV
was
composed
of
11
specimens
showing
eith
er
or
both
micro-
and
macronodular
cirrhosis,
with
Il
ePatocellular
regeneration,
bile-ductular
hyperplasia,
and
fi
brosis.
In
some
specimens
the
cirrhosis
was
in-
active,
whereas
in
others
there
was
a
coexistence
of
August
I,
1979
a)
73
CP
E
2
1
0.5
1
AGE
(YEARS)
1.5
2
Fig
9
—There
was
no
change
in
concentrations
of
hepatic
copper
in
repeat
liver
biopsy
specimens
in
three
unaffected
Bedlington
Terriers
(0).
In
contrast,
six
of
seven
affected
(•)
pups
had
marked
in-
creases
over
intervals
of
7
to
12
months
after
the
initial
assay.
chronic
active
hepatitis
with
cirrhosis,
ie,
ongoing
de-
generation
and
regeneration
as
well
as
necrosis
of
he-
patocytes
with
pronounced
inflammatory
responses.
Dark
granules
were
abundant.
One
specimen
was
al-
most
devoid
of
normal
parenchymal
cells
and
consisted
of
degenerated,
li
pid
-laden,
or
necrotic
hepatocytes
be-
tween
areas
of
fi
brosis
that
contained
proliferated
bile
ductules
and
zones
of
inflammation.
Occasionally,
di-
lated
lymphatics
were
seen
in
the
cirrhotic
specimens
(Fig
6).
Foci
of
extramedullary
hematopoiesis
were
common.
The
gross
appearance
by
peritoneoscopy
or
at
lapa-
rotomy
of
the
livers
in
groups
I
and
II
was
normal.
In
group
III,
the
lobes
of
the
li
ver
were
slightly
swollen,
with
rounded
edges,
and
the
surfaces
had
accentuated
lobular
outlines.
With
increasing
fi
brosis,
the
surface
of
the
liver
appeared
fi
nely
nodular.
When
cirrhosis
was
established
(group
IV),
the
liver
appeared
dark
brown,
with
a
mixed
fi
nely
and
coarsely
nodular
surface
(Fig
7).
Generally,
these
end
-stage
livers
were
of
normal
or
slightly
decreased
size.
Correlation
of
Hepatic
Injury
with
Copper
Concen-
tration
—The
copper
content
of
the
liver
and
the
number
and
prominence
of
the
cytoplasmic
granules
increased
with
age
of
affected
dogs
until
the
6th
year,
after
which
a
slow
decline
in
mean
copper
concentration
seemed
to
occur
(Fig
1
and
8).
In
sequential
biopsy
samples
obtained
over
6-
to
12
-month
intervals
on
three
unaffected
dogs
and
on
seven
affected
dogs
in
the
same
age
range,
the
hepatic
copper
concentration
was
un-
changed
in
the
normal
dogs
and
increased
by
more
than
500
p.g
of
copper/g
dry
tissue
in
six
of
the
seven
af-
fected
dogs
(Fig
9).
The
hepatic
injury
appeared
to
273
PIGMENT
GRANULES
90
1E3
FOCAL
HEPATITIS
I
I
80
0
CHRONIC
ACTIVE
HEPATITIS
III
70
CIRRHOSIS
Iv
60
50
40
:•:
30
20
10
FP:i•
0-I
1-2
2-4
4-6
6-8
8-10
>10
AGE
GROUPS
(YEARS)
Fig
10
—Changes
in
relative
frequncy
of
four
types
of
histologic
lesions
as
a
function
of
age
in
68
affected
Bedlington
Terriers.
The
bars
identified
as
"pigment
granules"
refer
to
group
I
of
the
histo-
logic
classification,
ie,
specimens
of
liver
with
pigment
granules
in
otherwise
uninjured
hepatocytes.
The
other
bars
refer
to
groups
II
to
IV,
with
progressive
signs
of
hepatic
injury
in
addition
to
pig-
ment
granules.
be
most
severe
in
the
oldest
dogs
and
in
those
with
the
greatest
copper
concentration
(Fig
10).
As
the
affected
dogs
grew
older,
hepatic
injury
continued
to
progress,
with
chronic
active
hepatitis
and
cirrhosis
the
predominant
lesions,
despite
the
slight
decrease
in
he-
patic
copper
concentrations
noticed
after
6
years
of
age.
Clinical
Signs
—In
only
19
of
the
68
affected
dogs
were
there
signs
of
li
ver
disease
at
the
time
of
biopsy
or,
indeed,
before
necropsy.
There
were
three
distinct
clinical
syndromes.
(1)
Acute
episodes
of
anorexia,
vomiting,
weakness,
lethargy,
and
dehydration
occurred
in
fi
ve
dogs
less
than
6
years
old.
With
only
supportive
therapy
all
recovered
within
a
few
weeks.
These
dogs
had
morpho-
logic
and
biochemical
evidence
of
active
liver
disease.
Precipitating
events
other
than
a
change
in
the
environ-
ment
in
one
dog could
not
be
identified.
(2)
In
13
older
dogs
(three,
5
to
8
years
old;
and
ten,
9
to
12
years
old),
insidious
deterioration
of
their
general
condition
(anorexia,
weight
loss,
intermittent
vomiting,
diarrhea,
and
a
general
unthriftiness)
was
noticed
for
weeks
to
months
before
jaundice,
ascites,
cachexia,
hepatic
encephalopathy,
and
death
supervened.
(3)
An
acute
onset
of
hemolytic
anemia,
and
jaun-
dice
markedly
increased
SGPT
activity
indicative
of
he-
patocellular
necrosis,
occurred
in
one
bitch
following
whelping
and
during
lactation.
The
dog
recovered
from
this
episode
but
died
6
months
later.
Three
of
the
13
dogs
in
the
second
group
also
had
hemolytic
episodes
preterminally.
In
only
fi
ve
of
the
affected
dogs
was
there
palpable
hepato-
or
splenomegaly.
None
of
the
10
dogs
in
which
slit
-lamp
biomicroscopic
examinations
were
performed
had
evidence
of
copper
deposits
in
the
cornea
that
con-
stitute
the
Kayser
-Fleischer
rings
so
characteristic
of
classic
inherited
copper
toxicosis
of
man
—Wilson's
disease.
All
19
dogs
with
clinical
signs
had
various
ob
nor,
malities
in
liver
function
and
almost
always
h
a
d
in:
creased
SGPT
activity.
Of
the
49
dogs
without
8
4
4
of
liver
disease,
only
22
had
abnormally
high
sort
activity
(ranging
from
71
to
mo,re
than
500
ru;
norn
itt
i
,
less
than
60
iu).
Clearly,
the
increase
in
SGPT
activity
occurred
relatively
late
and
reflected
hepatocellular
jury
rather
than
uncomplicated
copper
overload.
B
o
th
the
frequency
and
amount
of
the
increase
correlated
roughly
with
the
histopathologic
groupings.
Th
us,
i
n
group
I,
ie,
dogs
without
li
ght
microscopic
signs
o
f
liver
cell
injury,
only
8%
had
minimal
increases
i
n
SGPT
values,
and
these
were
considered
to
be
of
li
ttle
clinical
significance.
In
contrast,
50%
of
the
dogs
i
n
the
histopathologic
group
II
had
SGPT
values
that
ranged
from
71
to
350
iu
(mean,
169
ru),
89%
of
group
III
had
SGPT
values
of
108
to
500
ru
(mean,
250
ru),
and
all
of
the
dogs
in
group
IV
had
SGPT
values
ranging
from
120
to
500
m
(mean,
322
ru;
Fig
11).
Only
with
ad-
vanced
disease
did
the
alkaline
phosphatase
and
total
bilirubin
values
increase
and
the
concentrations
of
pro
-
thrombin
and
serum
albumin
decrease.
Serum
Copper
—There
was
no
significant
difference
between
mean
±
SD
of
serum
copper
concentrations
in
7
mixed
-breed
dogs
(49
±
5
j
Ag/d1),
15
affected
(54
±
10
µg/dl),
and
7
unaffected
Bedlington
Terriers
(56
±
10
p.g
/
dl)
Therapy
—Thirteen
affected
dogs
were
given
daily
doses
of
0.125
or
0.25
g
of
D-penicillaminee
about
30
minutes
before
a
meal.
This
chelating
agent
mobilizes
copper
from
tissues
and
promotes
its
excretion
in
the
urine.
Its
administration
has
produced
dramatic
ther-
apeutic
and
prophylactic
effects
in
patients
with
Wilson's
disease.
6
The
medication
was
discontinued
in
one
dog
because
of
vomiting.
None
of
the
toxic
manifestations
reported
in
man
has
been
observed
in
dogs
treated
for
as
long
as
3
years.
Genetics
—The
mating
of
two
affected
Bedlington
Terriers
resulted
in
a
litter
of
fi
ve
affected
pups.
Based
on
this
observation,
we
conclude
that
the
underlying
defect
in
copper
metabolism
is
genetic.
As
yet
we
are
unable
to
determine
the
prevalence
of
this
abnormalitY
since
most
of
the
Bedlington
Terriers
we
have
studied
were
referred
by
their
worried
owners,
leading
almost
certainly
to
a
much
higher
incidence
of
the
abnormal
gene
in
our
90
dogs
than
in
the
breed.
4
Discussion
The
concentration
of
hepatic
copper
in
many
of
the
affected
dogs
was
about
5
to
50
times
that
of
normal
dogs
or
100
to
400
times
that
of
normal
human
sub-
jects.'
Light
microscopic
evidence
of
liver
disease
has
been
observed
in
most
affected
terriers,
with
all
of
then)
having
distinctive,
dark,
intracytoplasmic
copper
-con:
taining
granules
in
the
hepatocytes.
These
lysosonaal
granules
appear
early
in
life
and
sequester
the
e
xcess
copper
progressively,
thereby
protecting
cytoplasmic
01
"
ganelles
from
copper
toxicity
until
their
capacity
for
copper
is
overwhelmed
and
hepatic
injury
ensues.
8
9
Cuprimine,
Merck
Sharp
&
Dohme,
West
Point,
Pa.
274
JAVMA,
Vol
175,
14
1
100
r
PIGMENT
FOCAL
CHRONIC
CIRRHOSIS
GRANULES
HEPATITIS
ACTIVE
HEPATITIS
NORMAL
SGPT
t
SGPT
Fig
1
1
—Relative
frequencies
of
normal
and
increased
serum
glutamic-
pyruvic
transaminase
activity
in
68
affected
Bedlington
Terriers
as
a
function
of
progression
of
the
histologic
classification
of
liver
injury.
I
The
activity
of
SGPT
was
frequently
increased
in
the
affected
dogs
(Fig
11),
even
when
clinically
normal,
al-
though
the
amount
of
the
increase
varied
considerably
as
a
function
of
the
severity
of
the
hepatocellular
injury.
Abnormalities
in
the
concentrations
of
plasma
alkaline
phosphatase,
bilirubin,
prothrombin,
and
albumin
were
more
common
in
overtly
ill
animals
but
were
of
no
specific
diagnostic
significance.
A
presumptive
diagnosis
of
copper
toxicosis
should
be
considered
in
any
Bed-
li
ngton
Terrier
with
persistently
high
SGPT
activity,
but
a
normal
SGPT
value
does
not
rule
out
this
diagnosis.
Both
quantitative
copper
analysis
and
li
ght
microscopic
examination
of
a
biopsy
or
necropsy
sample
of
liver
stained
with
hematoxylin
and
eosin
for
the
detection
of
abnormal
cytoplasmic
granules
in
the
hepatocytes
are
required
for
definitive
diagnosis.
From
our
experience,
by
using
the
two
procedures
together,
we
can
distinguish
affected
from
unaffected
dogs
that
are
as
young
as
6
months
(Fig
9).
We
have
not
established,
however,
whether
routine
histochemical
staining
for
copper
adds
to
the
accuracy
of
the
diagnosis.
Except
for
a
few
young
dogs
with
acute
and
fatal
hepatic
disease,
illness
was
manifested
only
in
dogs
with
advanced
hepatic
lesions.
From
our
li
mited
experience,
Penicillamine
appears
to
be
of
some
therapeutic
value.
It
seems
logical,
as
well,
to
restrict
the
supply
of
dietary
Copper
to
affected
dogs
by
avoiding
commercially
pre-
pared
dog
foods
inasmuch
as
all
contain
5
to
10
mg
copper/kg,
as
recommended
by
the
National
Research
Connell°
Therefore,
a
balanced
home
diet
that
ex-
cludes
few
foods
with
high
copper
contents
(eg,
liver,
brains,
and
sweetbreads)
and
the
omission
of
copper
-
S
upplemented
vitamin
-mineral
preparations
are
recom-
mended.
Copper
toxicosis
in
Bedlington
Terriers
is
remarkably
similar
to
human
Wilson's
disease.
9
In
the
latter,
which
la
transmitted
in
autosomal
recessive
fashion,
dietary
copper
accumulates
to
toxic
excess,
initially
in
the
liver,
probably
because
of
a
genetic
defect
in
a
normal
hepatic
lysosomal
mechanism
for
excreting
copper.
The
evolu-
tion
of
the
structural,
functional,
and
clinical
hepatic
abnormalities
that
are
produced
by
the
copper
resembles
that
seen
in
the
Bedlington
Terriers
of
this
report.
But
the
disorders
differ
in
at
least
two
respects.
First
extra
-
hepatic
copper
toxicosis—which,
except
for
hemolysis,
has
not
been
seen
in
the
dogs
—occurs
in
more
than
one
half
of
patients
with
Wilson's
disease,
most
disastrously
as
disturbances
of
central
nervous
function.
Second,
although
serum
concentrations
of
copper
and
of
the
copper
protein,
ceruloplasmin,
in
affected
Bedlington
Terriers
were
not
different
from
those
of
normal
dogs,
these
analyses
are
of
great
diagnostic
significance
in
Wilson's
disease,
wherein
the
concentration
of
noncerulo-
plasmin
plasma
copper
is
abnormally
high
and
that
of
ceruloplasmin
is
abnormally
low.
11
Unfortunately,
the
simple
enzymatic
method
of
determining
human
cerulo-
plasmin
concentration's
has
not
proved
adaptable
to
ca-
nine
serum.
A
further
search
for
involvement
of
the
central
nervous
system
in
affected
Bedlington
Terriers
and
the
development
of
an
accurate
assay
for
canine
serum
ceruloplasmin
will
be
required
before
we
can
say
how
congruent
a
model
the
copper
toxicosis
of
Bedling-
ton
Terriers
is
for
Wilson's
disease.
References
1.
Padula
M:
A
breeder's
experience
with
liver
malfunction.
Bull
Bedlington
Terrier
Club
Am,
Spring,
1974,
pp
11-16.
2.
Hardy
RM,
Stevens
JB,
Stowe
CM:
Chronic
progressive
hepatitis
in
Bedlington
Terriers
associated
with
elevated
liver
copper
concentrations.
Minn
Vet
15:13-24,
1975.
3.
Morell
AG,
Windsor
J,
Sternlieb
I,
et
al:
Spectroscopic
determinations
of
microgram
quantities
of
copper
in
biological
materials,
in
Sunderman
FW,
Sunderman
FW
Jr(ed):
Labora-
tory
Diagnosis
of
Liver
Diseases.
St
Louis,
WH
Green,
1968,
pp
196-198.
4.
Sipponen
P:
Orcein
positive
hepatocellular
material
in
long-standing
biliary
diseases.
I.
Histochemical
characteristics.
Scand
J
Gastroenterol
11:545-552,
1976.
5.
Sternlieb
I:
Mitochondria)
and
fatty
changes
in
hepato-
cytes
of
patients
with
Wilson's
disease.
Gastroenterology
55:
354-367,
1968.
6.
Sternlieb
I,
Scheinberg
IH:
Penicillamine
therapy
for
hepatolenticular
degeneration.
J
Am
Med
Assoc
189:748-754,
1964.
7.
Scheinberg
IH,
Sternlieb
I:
Metabolism
of
trace
metals,
in
Bondy
PK
(ed):
Duncan's
Diseases
of
Metabolism.
Philadel-
phia,
WB
Saunders
Co,
1969,
vol
2,
pp
1321-1334.
8.
Goldfischer
S,
Sternlieb
I:
Changes
in
the
distribution
of
hepatic
copper
in
relation
to
the
progression
of
Wilson's
disease
(hepatolenticular
degeneration).
Am
J
Pathol
53:883-901,
1968.
9.
Sternlieb
I:
Evolution
of
the
hepatic
lesion
in
Wilson's
disease
(hepatolenticular
degeneration),
in
Popper
H,
Schaffner
F(ed):
Progress
in
Liver
Diseases.
New
York,
Grune
&
Strat-
ton,
1972,
vol
IV,
pp
511-525.
10.
Nutrient
Requirements
of
Dogs.
Washington,
DC,
Na-
tional
Academy
of
Sciences,
Publication
No.
0-309-02315-7,
1974.
11.
Scheinberg
IH,
Gitlin
D:
Deficiency
of
ceruloplasmin
in
patients
with
hepatolenticular
degeneration
(Wilson's
disease).
Science
116:484-485,
1952.
12.
Morell
AG,
Windsor
J,
Sternlieb
I,
et
al:
Measurement
of
the
concentration
of
ceruloplasmin
in
serum
by
determination
of
its
oxidase
activity,
in
Sunderman
FW,
Sunderman
FW
Jr
(ed):
Laboratory
Diagnosis
of
Liver
Diseases.
St
Louis,
WH
Green,
1968,
pp
193-195.
44
9us+
I,
1979
275