Download The Elution of 51Cr from Labelled Leukocytes -a

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The
Elution
of
51Cr
from
-a
New
By
T
HE
ELUTION
of
the
hampered
relatively
24 hours1
slow rate
of
) is undesirable
14C-glycine
elution
cells,
are
and
30-60
tein.6
Studies
cent
red
it has
been
elution
results
tein.
tein
However,
is such
that
moved
by
the stability
non
the
form
by postulating
chain
peptides
a postulate
its interesting
that
over
that
Peritoneal
cells
199
199
fresh
activity
F.C.S.
50-150
(B.S.S.)
for
four
and
dialysate
used
10
and
per
The
was
cells
removed
of
hexavalent
life
span.2
The
bound
to amino
groups
to proof lysine
chromium
is free
20
cellular
pro-
difficult
therefore
the rapid
elution
This
to reconcile
of 51Cr in a
difficulty
may
be
re-
the cell attached
to amino
acids
or
of labelled
cellular
protein.
Such
previously
in the
AND
advanced
and,
studies
reported
because
here.
of
METHODS
C57B1/6J
serum
and
chromium,
labelled
from
(199
mice
F.C.S.).
by
The
peritoneal
cells
were
lavage
with
resuspended
in
51CrO4
(specific
The cells were washed
three times with a balanced
salt solution10
in 10 ml. B.S.S. The resulting
suspension
was incubated
at 37 C.
were
then
sedimented
by centrifugation
at 350 g. for 7 minutes.
The
and
dialysed
against
distilled
water
for 48 hours
at 0-4 C. The
for
was then
concentrated
for further
study and will
A solution
calf
cell
its
per
and
rapid
than
from
red
reported
for leuko-
predominantly
leukocytes.
S
from
fetal
1 per cent
that
DF32P
cells.4’5
It seems
bond
with
labelled
obtained
cent
has
Even
bond
between
chromium
and pro51Cr bound
to albumin
could
be re-
to have
been
was investigated
incubated
Ci/Gm.).
resuspended
hours.
supernatant
were
+
red
chromium
that 51Cr is lost from
resulting
from
turnover
MATERIAL
Medium
eluted
of
30 days.8
from
cells
studies.
cells is more
having
been
it is bound
of the covalent
1 per cent
of
does
not seem
implications,
labelled
in the protein
by a firm covalent
bond.
and is not precipitated
by trichloracetic
chromium-protein
protein-bound
solved
short
measuring
for tumor
dissociation
in vitro
the
from
quantitative
is found
suggest
stability
less than
dialysis
of
24 hours
assumed
from
for
leukocytes
acid residues
is dialysable
widely
that
51Cr
and tumor
per 24 hours
per
cells7
and other
basic
amino
Since
eluted
chromium
acid,9
than
labelled
with
( 51Cr)
for
from
labelled
red cells
( about
has led to the demonstration
tracers
per
R0NAT
radionucide
leukocytes
per cent
within
Chromium
M.
chromium
this
elution
and
better
of 51Cr from
rates
of 50-60
cytes’4
of
Leukocytes
Theory
PETER
radioactive
use
Labelled
1 hour
by
at 37
evaporation
henceforth
51Cr
(5iCr
C.
at
be cabled
VI)
with
was
250
37
C.
jzCi
overnight.
“eluted
prepared
51Cr
as
Na
This
concentrate
was
51Cr.”
by
dissolving
10
jCi
51Cr
as
Na2
From
tile Departments
of Medicine
(Nuclear
Medicine,)
and Pathology,
University
of
Sydney,
Sydney,
N. S. W., Australia.
First submitted
June 28, 1968; accepted
for publication
October
3 1968.
PETER
M. R0NA5,
MB.
B.S.,
B.Sc.
(M.),
PH.D.:
Present
address:
Division
of
Nuclear
Medicine,
The
Institute
of Medical
and
Veterinary
Science,
Royal
Adelaide
Hospital,
408
Adelaide,
South
Australia.
BLOOD,
VOL.
33, No. 3
(MARCH)
1969
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409
ELUTION
OF
51
51C#{216}4in
1 ml. B.S.S.
A further
aliquot
ion IIC1 and absolute
ethanol11
HC1 and ethanol
were removed
as
51Cr-labelled
above
) in
One
gram
was
dissolved
hire
only
was
at
by
lysine
1 ml.
the
bovine
in
dialysed
the
ml.
of
10
of
jCi
to
prepared
12
mgm.
water
lysine
51Cr
by
dissolving
either
at
and
III
1 :250
37
for
12
arginine.12
(prepared
performed
was
treated
as
with
bysine
hours.
The
)
above
jCi
or
with
excess
( 51Cr III ) The
.
in 1 ml.
51Cr
III
1 mg.
B.S.S.
(prepared
arginine.
Serum
Laboratories,
Melbourne)
( Difco ) were added.
The mix-
trypsin
C.
10
1 mg
( Commonwealth
and
distilled
as Na2 51CrO4
51Cr VI to the trivalent
state
and the 5tCr III dissolved
containing
albumin
were
51Cr
were
B.S.S.
B.S.S.
groups
manipulations
of
of
against
carboxyl
adsorption
arginine
plasma
20
addition
All
and
volumes
of 10 jzCi 51Cr
to reduce
the
by evaporation
disposable
Trypsin
resultant
to
the
hydrolyses
peptides
protein
were
labelled
dialysate.
plastic
equipment
to
avoid
the
glass.
51Cr VI, 51Cr III, 51Cr-lysine,
51Cr-arginine,
51Cr-peptides
and eluted
51Cr were cornpared
by electrophoresis
on paper
at 11OV using Verona!
buffer pH 8.6. Electrophoretograms
were cut into 1 cm. strips which
were counted
in a Packard
Autogamma
automatic
scintillation counter
using pulse height
analysis
to count only at the 323 keV photopeak
of 51Cr.
In
ed
another
experiment,
intravenously
counting
into
using
Decade
two
51Cr
VI,
51Cr
mice
and
24
a modified
Nuclear
III,
51Cr-peptides
hour
whole
Chicago
well
and
body
eluted
retention
counter
51Cr
were
determined
and
Nuclear
each
by
inject-
whole
Chicago
body
Model
186
Scaler.
RESULTS
Electrophoresis
A
graph
of
of the
the
various
distributions
of
51Cr
preparations
51Cr
activity
shown
in Figure
51Cr
towards
VI is not shown
since,
by 12 hours,
the anode.
(Shorter
electrophoretic
conducted
for 12 hours.
12 hours
electrophoresis
is
was
after
1.
it had migrated
runs confirmed
right
off the paper
this anodal
migra-
tion.)
51Cr
haps
III remained
the
at
origin
of binding
as a result
51Cr-lysine
and 51Cr-arginine
acids are on the positive
side
51Cr-peptides
showed
at
runs
5 and
also
After
51Cr
9 cm.
confirmed
intravenous
preparations
all electrophoretic
experiments,
per-
paper.
migrated
towards
the cathode.
(These
of their isoelectric
points
at pH 8.613).
a peak
5 and 9 cm. on the anodal
side
“Eluted
51Cr” also showed
peaks
during
to the
at the
origin,
of the origin.
a peak
at
on the anodal
side
the absence
of 51Cr
injection
into
at 24 hours
mice,
were
and
the
at least
origin,
and
two
at
further
least
amino
peaks
two
at
further
of the origin.
(Shorter
electrophoretic
VI in the eluted
51Cr preparation.)
whole
body
retention
of the respective
as follows:-
51Cr
VI
:
51Cr
III
:
41 per cent
91 per cent
51Cr-peptides
:
5 per
cent
eluted
:
4 per
cent
51Cr
DIscussioN
The
electroplioretic
labelled
peptides
and
hexavalent
or trivalent),
The
peptides,
behavior
biologic
both
being
behavior
of eluted
is not characteristic
nor of the behavior
of eluted
almost
51Cr
completely
is also
51Cr is very
similar
to that
of 51Crof the behavior
of free 51 Cr (either
of 51Cr-labelled
lysine
or arginine.
very
excreted
similar
within
to that
of 51Cr-labelled
24 hours
after
intra-
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RONAI
410
ELECTROPHORESIS
FOR
12
HOURS
12000
51C’
FlU
III
I
U
I
V
I
V
CELLS
I
U
‘0’
A
Fig. 1.-Twelve
hour
electrophoretogram
of free
51Cr III, 51Cr-labelled
lysine,
51Cr-labelled
arginine,
51Cr-labebled
peptides
and eluted
51Cr. (By 12 hours
51Cr
VI has migrated
right
off the paper
towards
the anode
and is not shown
here.)
The
labelled
peptides
show
electrophoretic
behavior
very
similar
to that
of eluted
51Cr
with peaks
at 0, 5 and 9 cm.
The origin
(0) is marked
with an arrow,
the cathode
is to the left and the anode
to the
right.
The
figures
on the
abscissa
This
contrasts
represent
1 cm.
strips
on the electrophoresis
paper.
venous
injection.
valent
51Cr,
virtue
of their
both
of which
ability
Although
eluted
phoretic
peak
at
the
to label
51Cr and
origin,
with
are
cells
largely
and
51Cr-labelled
the whole
the
behavior
retained
protein
body
of
after
(respectively)
free
hexavalent
intravenous
and
injection
triby
in vivo.
peptides
both
showed
an electrocounting
results
show
that
this
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ELUTION
411
51cii
OF
Table
l.-51Cr
activity
in supernatant
at intervals
during
in vitro
incubation
51Cr-labelled
mouse
peritoneal cells in a balanced
salt solution
at 37 C. Counts
the supematant
are divided
into protein-bound
(determined
by trichioracetic
acid
precipitation)
and non protein-bound
fractions.
5 mm.
Total
(c.p.rn.)
Protein-Bound
Activity
is possible
extracellular
lytic
enzymes,
unlikely
3 hr.
4 hr.
8,535
19,457
21,320
25,869
that
917
14.5%
1,121
15.3%
720
9.2%
1,207
14.1%
2,869
14.7%
2,511
11.8%
3,725
14.4%
5,399
85.5%
6,192
84.7%
7,105
90.8%
7,328
85.9%
16,588
85.3%
18,809
88.2%
22,144
85.6%
7,825
as
6-10
there
the
times
of the
the
as
of
these
leukocytes
in
of
much
non
findings,
Further,
their
by
loss
is suggested
the
body.
These
conclusions
the
from
apply
to
turnover
cell
mouse
and
the
above
hypothesis
powerful
technic
metabolism.
termined
by
for
For example,
measuring
injection
study
the
between
cells
activity
in
counts
show
peritoneal
the
elution
rapid
cells,
of
proteins
there
the
superthat
cell
51Cr
complete
but
as
from
is no great
Whether
cannot
the
cata-
excretion
there
in general.
in erythrocytes
from
within
to interrupt
the
are non-reutilisable
correct,
would
it
of both
seem
intracellular
to
provide
and
con-
or not the
of course
the assumption
of the labelled
plasma
that
protein.
proteins.
labelling
Such
with
protein
could
be deafter
the intra-
investigations
51Cr
does
but
a simple
extracellular
the turnover
rate
of plasma
proteins
rate
at which
activity
is excreted
of 51Cr-labelled
ever,
depend
on
rate of catabolism
that
the
hydroseems
experiments.
is
the
)
labelled
labelled
appears
peptides
their
difficulty
in applying
them
to leukocytes
mechanism
of chromium
elution
is the same
from
that
of
ceptual
be determined
If the above
cell
from
cellular
This
1
of the
protein-bound
The
binding
of chromium
to the protein
process
at the peptide
level.
The resulting
evidenced
result
( Table
shows
peptides
for 51Cr elution.
it
from
supernatant
incubation
activity.
to account
results
the
51Cr-labelled
cellular
protein
and
by cell disintegration.
medium
start
is protein-bound
is insufficient
light
labelled
after
represents
peptides
51Cr-labelled
been
released
sampling
4 hours
disintegration
In
7,313
51Cr-labelled
periodic
and
is consistently
natant
6,316
free 51Cr III. It probably
point
at pH 8.6.
interaction
of
both
having
since
5 minutes
venous
2 hr.
Supernatant
cannot
be due to
with an isoelectric
the
1 hr.
Supernatant
(c.p.rn.)
% of Total
Non Protein-Bound
Activity
(c.p.rn.)
% of Total
cell.
bolic
30 mm.
Supematant
Activity
It
15 mm.
of
in
do,
not
howthe
alter
SUMMARY
in
Chromium-51
vitro
and
trichioracetic
in
acid
elutes
vivo.
and
rapidly
Eluted
it has
from
labelled
leukocytes
51Cr is dialysable
and
therefore
been
assumed
and tumor
cells
is not precipitated
to
be
free
both
with
chromium
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412
RONAI
which
has dissociated
from
the labelled
cellular
protein.
In vitro
studies
with
51Cr-labelled
albumin
have,
however,
demonstrated
the extreme
stability
of
the covalent
bond
between
chromium
and protein.
Paper
electrophoresis
experiments
are here
described
which
show
that the
51Cr
which
ally
like
elutes
from
labelled
51Cr-labelled
or trivalent
Whole
and
forms,
or 51Cr-labelled
body
counting
studies
51Cr
preparations
also
have
the
presence
the 51Cr
results
cells
behaves
free
51Cr
that
and
eluted
whole
injection
51Cr
body
electrophoretic-
in either
and
hexavalent
of the
various
51Cr-labelled
counting
peptides
studies
exclude
the
amounts
of free 51Cr ( either
hexavalent
or trivalent
) in
from
labelled
cells and supports
the hypothesis
that 51Cr
turnover
of labelled
protein
within
the cell and loss of
from
non-reutilisable
at all like
lysine
and arginine.
after
the intravenous
electrophoretic
of significant
that elutes
labelled
Confirmation
of intra
and
peritoneal
not
into mice indicate
biologic
behavior.
similar
Together,
elution
mouse
peptides
protein
of this
extracellular
fragments.
theory
would
provide
protein
metabolism.
a powerful
technic
for
the
study
ACKNOWLEDGMENTS
This
study
Doctor
of
Health
and
was
performed
in
Philosophy
at
Medical
Research
University
of
Sydney
the
partial
fulfilment
Council,
Cancer
the
Research
New
e-per
consequente-il
ha
proteina
cellular.
Tarnen,
extreme
stabilitate
del
Es
describite
que
es
corno
ab
peptidas
trivalente
de
51Cr
mente
simile
Le
studios
in
51Cr
resulta
cate
fragmentos
de
de
proteina
the
ilbo
albumina
Cancer
Council
State
con
post
eluite
es chromo
libere
marcate
and
the
tanto
in vitro
trichboracetic,
dissociate
a 51Cr
ad
ha
be marcate
demonstrate
be
e proteina.
a papiero
del toto
total
51Cr
of
National
acknowledged.
chromo
de
marcate
degree
of the
be quales
muses
se
como
51Cr
monstra
comporta
que
be 51Cr
electrophoreticarnente
libere
in
forma
hexavalente
o
51Cr.
be injection
e eluite
intravenose
peptidas
del
marcate
comportamento
biologic.
insimul
con be studios de contation
vane
con
preparatos
51Cr
ha
equal-
de
de
quantitates
de
iste
significative
de
a corpore
libere
51Cr
total
exclude
(hexavalente
o
be
triva-
ad cellulas
marcate
e supporta
be hypothese
que le elution
de
de proteina
marcate
intra be celluba
e ab perditas
de mar-
non-reutilisabile
confirmation
olismo
que
que es eluite
be metabolismo
ab
que
peritoneal
corpore
indica
presentia
be 51Cr
lente)
Wales
gratefully
con
inter
5mCr e non
electrophoretic
de
for
support
INTERLINGUA
electrophorese
e arginina
characteristicas
possibilitate
Le
con
del
muses
vitro
covalente
con
lysina
in
in
cellulas
contation
ad
supponite
bigamine
marcate
o corno
Studios
con
essite
studios
marcate
is
IN
requirements
generous
eluite
ab marcate
leucocytos
e cellulas
turnoral
es dialysabile
e non es precipitate
con acido
experimentos
eluite
the
The
South
Fund
SUMMARIO
Chromo-51
es rapidemente
como
in vivo. Le 51Cr eluite
of
of Sydney.
University
theoria
intracellular
proteina.
providerea
un
potente
technica
pro
Ic
studio
del
metab-
e extracelbular.
REFERENCES
1. Necheles,
Leroy,
for
J.
C.
the
Lab.
2.
T. F.,
V.:
Radioactive
study
Clin.
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Weinstein,
of
survival
Med.
M.
J.,
42:358,
and
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of
red
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Berlin, N.
evaluation
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of
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Blood
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An
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red
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as
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A.:
Con-
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B.,
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la stabilit#{233} du
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marquage
in vitro
des
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ELUTION
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413
leucocytes
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le P-32, le Cr-51
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Franc.
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Han,
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9. Anghileri,
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11. Vogel,
1966.
5. Vincent,
P. C., and Nicholls,
A.: The
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radiation
damage
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Canad.
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Invest.
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of
with
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red
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blood
radioactive
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amides
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pro-
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1950.
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of
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1967,
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p.
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(ed.
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complexes.
Elliott,
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The
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ef-
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926.
13.
Ei-
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amino
Myrkack,
K.
Sutherland,
and
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520.
Chemistry
The
plasma
chromium.
sentraut, A. M., and
Lanz,
tagging
of beukaemic
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in vivo cell
717, 1955.
K.:
p.
12. Zittle, C.
p.
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tagging
teins
1961,
staalbu-
stimulation
on incorporaand methionine
into procells.
J. Exp. Med.
110:
A. I.: A Textbook
Analysis.
London,
Inorganic
Part
1964.
7.
tive
of the
serum
Data
2).
Oxford
Stability
Dawson,
W.
for
H.,
constants
H.
and
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University
M.
C.,
Jones,
RePress,
430.
14. Ronai,
P. M.: Studies
on the rejection
of radioisotope-labelled
grafts.
Ph.D.
Thesis,
University of Sydney,
1967.
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
1969 33: 408-413
The Elution of 51Cr from Labelled Leukocytes −−a New Theory
PETER M. RONAI
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