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Lymphocyte
By
and
L.
HENRY
Granulocyte
with Down’s
NADLER,
UMEROUS
in the
Down’s
syndrome.’
activity
is
leukocyte
in
blood
cells
sults
have
the technic
rophosphate
mass,
upon
syndrome
with
hand,
higher
trisomic
locyte
In an
responsible
utilized
Engel2
published
age
enzyme
Toimu
demonstrating
and
levels.
among
suggested
Two
patients
that
that
result
IN0UYI:
HsIA
enzymes
have
in Patients
a
the
presence
recent
studies
increase
trisomic
level
shortened
in the
an
with
of enzyme
lifespan
of
of younger
with
the
white
conflicting
re-
to
)
answer
this question.
Gaibraith
and Valberg,3
using
leukocytes
in vitro
with
radioactive
diisopropyl
fiuoto study
the granubocyte
half-life,
the blood
granulocyte
turnover
and
et al.,4
using
syndrome,
rate
four
were
patients
comparable
were
unable
with
technics
able
to
to
Down’s
demonstrate
involving
demonstrate
any
syndrome.
On
DFP32
an
increase
the
labeling
in
granu-
rate.
attempt
for
granulocytes
cell
might
normals
Down’s
turnover
been
cell
granulocyte
Raab
have
Activity
MONTELEONE,
YI-YUNG
blood
and
Down’s
between
other
have
Hook
dependent
the
difference
in
of white
attempted
of labeling
( DFP32
and
DAVID
REPORTS
activity
L.
PATRICIA
AND
N
Enzyme
Syndrome
the
in
to demonstrate
the elevation
method
Garvin’s
in
which
white
blood
enzyme
activities
of Rabinowitz5
glass
bead
in the
blood
( C),
of lymphocytes
and
AND
was
who
METHODS
obtained
from 10 patients
with trisomic
Down’s
are residents
at the Dixon State School, Dixon,
and sex and had an age range
of 6 to 10 years.
30 minutes
with 6 per cent dextran
in normal
saline
(Abbott)
mixed
in a ratio of 1 ml. of dextran
per 5 ml. of blood.
The supernatant
was removed
to within
0.2 cm. of the red blood
cells and centrifuged
at 100 g for 10
minutes.
The white
cell pellet
was
then resuspended
in 4 ml. of serum.
The remainder
of
the supernatant
was centrifuged
at 1500 g for 10 minutes
and this cell-free
serum
used for
both
the preparation
procedure
and the elution
of the column.
The general
procedure
of preparation
of the column
and cells and the actual
separation
The
blood
was
sedimented
for
separation
be
we
columns.6
MATERIALS
Twenty-five
ml. of heparinized
syndrome
( M ) and 10 controls
Ill. The two groups
were matched
cell component
might
in Down’s
syndrome,
age
at 37 C. for
From
the Genetic
Clinic
of the Children’s
Memorial
Hospital
and the Department
of
Pediatrics,
Northwestern
University
Medical
School,
Chicago,
Ill.
These
studies
were
aided
by grants
from
the Otlao S. A. Sprague
Memorial
Fund,
the
Illinois
Mental
Fund,
and the U. S. Public
Health
Service
(1-SOL-FR
5070)
(1-SOLFR
5475)
(T1-AM-5186).
submitted
March 2, 1967; accepted
for publication
April
13, 1967.
HENRY
L. NADLER,
M.D.:
Associate
in Pediatrics,
Northwestern
University,
Children’s
Memorial
Hospital,
Chicago,
ill. PATRIcIA
L. MONTELEONE,
M.D.:
Instructor
in Pediatrics,
Northwestern
University,
Children’s
Memorial
Hospital,
Chicago,
Ill. Tomiu IN0uYE,
PH.D.:
Assistant
Professor
of Biochemistry,
University
of Illinois,
Chicago,
Ill. DAVID
YI-YUNC
H5IA,
M.D.:
Professor
of Pediatrics,
Northwestern
University,
Children’s
Memorial
Hospital,
Chicago,
Ill.
First
669
BLOOD,
VOL.
30,
No.
5
(NOVEMBER),
1967
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670
NADLER
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I
IQtI
I
I.
I’?
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t.o
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I..
U
1+1+1+1
U
I
v
II
- V
INtr)c
I+1+1+,
2
IO\t
H-
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00
=
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I+i+i+i
Isr’i
-
I
-
I
z
<L
I
H
ET
AL.
From www.bloodjournal.org by guest on August 9, 2017. For personal use only.
LYMPHOCYTE
AND
GRANULOCYTE
ENZYME
671
ACFIVITY
of cells were those of Rabinowitz.5
Mir’imal
essential
media
modified
for suspension
culture
(G. I. B. Co. ) was substituted
for Hanks BSS. In addition,
the column
was incubated
at
37 C. for 20 minutes
instead
of 30 minutes.
Plastic
tubes,
pipettes,
and siliconized
glassware were
used throughout.
A sample
of the sedimented
white
blood
cells
( W)
was placed
on the column,
the
lymphocyte
fraction
( L ) was obtained
by elution
with
100 per cent cell-free
serum, and
the polymorphonuclear
leukocyte
fraction
( P) was
obtained
by elution
with
EDTA
solution
( G. I. B. Co. ). Aliquots
of W, L and P were used for microscopic
analysis,
cell
count
( using
a Coulter
counter
) , and
enzyme
studies.
Acid
phosphatase
( AcP ) was
assayed
at pH 4.90
and alkaline
phosphatase#{176}
(AkP)
at pH 9.3, using
p-nitrophenol
phosphate
as substrate.7
Glucose-6-phosphate
dehydrogenase
( C-6-PD)
was
determined
spectroscopically
by the reduction
of NADP
at 340 m, using
C-6-P
as substrate.’
Separation
of white
cells was always
performed
in pairs
with one C and one M being
studied
simultaneously.
The column
was considered
satisfactory
if 50 per cent of P and
50 per cent of L were recovered
from W in both columns.
Approximately
70 per cent of
the columns
filled these criteria
and were
Included
in the results.
The L fraction
contamed
97 per cent lymphocytes,
3 per cent polymorphonuclear
leukocytes,
and was contaminated
by 2 to 3 red blood
cells per lymphocyte.
The P fraction
contained
95 per cent
polymorphonuclear
leukocytes
and 5 per cent lymphocytes
and monocytes.
The cell fractions
were
ith
the
treated
with
results
of
hypotonic
the
saline
enzyme
to
remove
any
red
cells
which
might
interfere
assay.
RESULTS
As
test
shown
in Table
1, significant
could
be found
between
C and
differences
D for W,
measured
by the Student
L, and P in all instances.
“T”
DIscussIoN
Several
eral
have
explanations
blood
of
suggested
“structural”
have
patients
that
genes
for
ed that
the extra
for certain
specific
“C”
localizing
a specific
with
The
most
demonstrate
and
activity
Nadler
white
changes
in the
authors12”3
to
periph-
of investigators911
might
contain
contain
evidence
enzyme
that
blood
cell,
been convincingly
proved.
labels
the neutrophilic
al.19
of fibroblasts
of AcP,
an
suggesting
of the
and
et
Other
might
date,
no
for
the
A number
chromosome
enzymes.
locus
lifespan
Valberg3
in
were
patients
with
Down’s
syndrome.
A number
of different
enzymes
red blood
cells8’’13’15’1#{176} of patients
tivity
to explain
chromosome
As of this
hypothesis
a shortened
Galbraith
specific
genetic
age
leukocyte,
has not
DFP32
specifically
while
offered
have
the
suggest-
the “regulator”
genes
has been
presented
the
extra
chromosome
mongolism.
recent
to the
certain
group
enzymes.
associated
is related
been
with
Down’s
syndrome.
the
extra
“G” group
have
derived
AkP,
and
failed
from
C-fl-PD
4 of
unable
have
with
to
the
in both
of enzyme
the
Cartwright
series.
Raab
6 patients
activity
polymorphonuclear
et al.14 has shown
that
et al.4 were
able
to
with
Down’s
to demonstrate
any
syndrome,
difference
been
reported
to be increased
Down’s
syndrome.
DeMars,17
demonstrate
trisomic
alteration
specifically
any
mongols.
the
W
difference
The
and
the
#{176}0.1
ml. of 0.3M magnesium
chloride
is required
for the P fraction,
threefold
increase
of AkP activity.
Neither
magnesium
chloride
nor
effect
upon
AkP activity
in the W or L fractions.
in 4
in the
Cox,18
in enzyme
finding
L and
of
ac-
increased
P fractions
in
since this results in a
zinc chloride
has any
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672
NADLER
this
study
is not
demonstrates
localized
related
to
cells
some
which
The
that
the
to any
particular
specific,
though
may
be
distribution
alteration
in
fraction.
white
blood
It would
appear
generalized,
characteristic
of
of enzyme
cell
Down’s
activities
of
predominantly
terest
to
The
that
requirement
in the
with
in
note
An
various
The
Mg+
specific
blood
genetic,
be
be related
increase
circulatory
is
blood
cell
beads
is of
in-
chloride.
loss
or to the
different
is in
It
magnesium
to an intracellular
is that
fractions
that the activity
has been
shown
to
technics.21’22
P require
glass
possibility
the
procedure
valuable
metabolic,
the
to the
activity
this
of Mg+
elution
isoenzymes
+
ion
procedure
are
present
components.
of
a
histochemical
separation,
+ may
alternative
cell
numerous
of adhering
application
to
cell
process
EDTA.
prove
P by
for
active
the
in
after
AL.
syndrome.
in the
accord
with
previous
studies.
Rabinowitz2#{176} has demonstrated
of G-6-PD
is four times
greater
in P than
L. AkP activity
reside
enzyme
that
phenomenon
ET
adjunct
and
of
to
clinical
white
the
blood
standard
cell
separation
methods
used
should
in
studying
problems.
SUMMARY
Patients
with
increases
of acid
dehydrogenase
arated
from
in enzyme
trisomic
in
white
activities
chromosome
Down’s
syndrome
phosphatase,
causing
both
blood
alkaline
lymphocytes
cells by
appears
Esseva
trovate
to be
que
patientes
causa
de phosphatase
directly
related
le syndrome
de
have
significant
glucose-6-phosphate
leukocytes
sepThe alteration
to genes
con
IN
located
on the
le
INTERLINGUA
tnisomic
syndrome
acide,
de
de phosphatase
alcalin,
cosa-6-phosphato
in le lymphocytos
e etiam in le leucocytos
ab le altere
leucocytos
per le procedimento
de Rabinowitz.
activitate
enzymatic
non es directemente
relationate
con genes
(jtIC
nivellos
and
syndrome.
SUMMARIO
elevate
to
found
and
polymorphonuclear
procedure
of Rabinowitz.
the
not
Down’s
were
phosphatase,
Down
ha
significativemente
e de dehydrogenase
de glupolymorphonucleari
separate
Ii pare
que Ic alteration
del
locate
super
Ic chromosomas
Down.
ACKNOWLEDGMENTS
The authors
wish to express
their appreciation
to Drs.
James
Carvin
for sharing
with us their experience
with
for permitting
us to study
the patients,
and to Elvira
Pribila,
and Bonnie
Williams
for technical
assistance.
Yale Rabinowitz,
Fritz Bach,
and
the columns,
to Dr. Paul Tillman
Kavaliunas,
Janet
Pi, Rosemary
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From www.bloodjournal.org by guest on August 9, 2017. For personal use only.
1967 30: 669-673
Lymphocyte and Granulocyte Enzyme Activity in Patients with Down's
Syndrome
HENRY L. NADLER, PATRICIA L. MONTELEONE, TOHRU INOUYE and DAVID YI-YUNG HSIA
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