[CANCER RESEARCH 34, 2957—2960,November 1974J
Effects ofBuffers and pH on in Vitro Binding of 67Gaby L1210
Leukemic Cells'
Jerry
D. Glickson,
John
Diviiion ofHematology,
Webb,2
and Richard
Department ofMedicine,
A. Gams
and the Cancer Research and Training Center, University ofAlabama in Birmingham School of
Medicine, Birmingham,Alabama 35294
@
SUMMARY
MATERIALS AND METHODS
The effect of sodium nitrate and a series of buffers on in
vitro 67Ga binding to L1210 leukemic cells at pH 6.8 ±0.2
and 37° at concentrations of iO@ to l02
M has been
investigated. The relative ability of these agents to inhibit
cellular incorporation of 67Ga is ethylenediaminetetraacetate
nitrate
(New
England
Nuclear,
North
Billirica,
Mass.)
containing 2 mCi/ml at noon of the day of delivery were used
within 2 weeks of purchase (t½
78.1 hr). Commercial
>
mg/nil.
nitrilotriacetic
acid
>>
citrate
>>>
bicarbonate
>
Buffer Inhibition. Stock solutions
preparations
of the citrate salt contained
The nitrate sample was approximately
sodium citrate, 2
0.5 M in nitrate.
phosphate, lactate, Tris > morpholinopropane
sulfonic acid
(MOPS), N-2-hydroxyethylpiperazine-N'-2-ethane
sulfonic
acid, nitrate
0. Inhibition probably results from formation
Dilutions were made in 0.9% NaC1 solution and counted in a
Model 1185 automatic well scintillation counter (Nuclear
Chicago Corp., Des Planes, 111.),that had been calibrated with
tumor membrane or which compete with intracellular receptor
complexes. However, direct interaction of buffers with the cell
membrane or with gallium (III) receptors, as well as effects of
investigation included sodium bicarbonate (J. T. Baker
Chemical Co., Phfflipsburg, N. J.); potassium dihydrogen
phosphate (American Chemical Society certified; Fisher
Scientific Co., Fair Lawn, N. J.); lactic acid (American
ofgallium
(III)complexes
which
areeither
impermeable
tothe
6 7Ga
buffers
on cellular
monotonic
metabolism,
have not been excluded.
decrease in the cellular incorporation
A
of 67Ga
samples
Chemical
occurs
between
pH6.2and7.8inthepresence
oftheinert Norwood,
buffer, lO2 M morpholinopropane
sulfonic acid.
Preferential
6 7Ga
Society
activity.
reagent;
Buffers
and salts used in this
Matheson,
Coleman,
and
Bell,
Co., St. Louis, Mo.); HEPES (Sigma); sodium nitrate
(Baker; analyzed reagent); tnsodium citrate (Fisher; American
Chemical Society certified), EDTA disodium salt (Cambridge
Chemical Products, Inc., Detroit, Mich.), and NTA (Sigma).
INTRODUCTION
tumors
clinical
of known
Ohio); Tris (Fisher Scientific Co.; certified primary
standard); morpholinopropane
sulfonic acid (MOPS) (Sigma
Chemical
Except
localization of 67Ga in a broad range of solid
and malignant lymphomas
has been employed in the
detection
and early staging of malignant disease by
scintigra@hy
(4,
1 1).
An
understanding
of
the
detailed
for modifications
6 7Ga-binding
procedure
described
described
in
below,
detail
the
in vitro
elsewhere
(8)
was
used. L12l0 cells were harvested from the peritoneal cavity of
female
C57BL X DBA/2
F1 (hereafter
called BD2F1 ) mice 6
mechanism of 7Ga localization in tumor cells may lead to the
rational development of optimum methods for the detection
days after i.p. inoculation of i0@ leukemic cells. The cells were
washed 3 times with 0.9% NaCl solution, and a 4% packed-cell
differences between normal and transformed cells that
determine their interaction with metal ions. To help attain
these objectives, an in vitro technique for measuring binding of
in an ice bath. Samples of the buffer or salt (1 ml)
volume
ofmalignant
neoplasms
andmayalsoreveal
some
underlying
stored
6 7 Ga
by
dispersed
cells
was
recently
developed
in
this
laboratory (8). Preliminary experiments indicated that a
number of buffers (bicarbonate, phosphate, Tris, barbital,
maleic acid, and citrate) inhibit in vitro uptake of 67Ga by
Ll 2 10 leukemic cells. This study examines buffer inhibition
and pH effects in more detail. The need to study the effects of
counterions on the incorporation of 67Ga by isolated tumor
cells is underscored by recent experiments by Konikowski et
aL (10), who report a significant difference in tumor
localization and clearance of the citrate, lactate, and chloride
@
6 ‘Gacitrate and 6 7Ga
salts of 67Ga as opposed
to the DTPA
salt.
(2.8
adjusted
I This
research
2 Present
was
address:
supported
Research
by
School
Grant
of
CA-13148
Chemistry,
University, P. 0. Box 4, Canberra, Australia 2600.
from
Australian
the
NIH.
pH
6.8
± 0.2
suspension
with
was prepared
HC1 and/or
NaOH
and
were
incubated in 12- x 75-mm Falcon 2052 counting tubes
(Becton, Dickinson and Company, Oxnard, Calif.) for 1 hr at
37
0
.
with 2.60 X 10
6 “Ga(delivered
in
—8
jimole
.
(1.05 jiCi, 1.5 X 10
100 jzl of0.9%
NaC1
solution).
At
6
cpm) of
specified
timeintervals,
250-jzl
aliquots
ofcellsuspension
wereadded
to
duplicate tubes and the incubation was continued. The
reaction was quenched by transferring the tubes to an ice bath
and adding 2 ml of iced 0.9% NaCl solution
cells were washed 3 additional times
(at 4°),and the residual radioactivity
pH Studies. Cell-binding studies
scribed above, with the use of
consisting
ReceivedMay 13, 1974; accepted July 16, 1974.
to
X l0@ cells/ml)
to each tube. The
with 0.9% NaCl solution
was determined.
were performed as de-•
an incubation medium
of 1 ml 10 2 M morpholinopropane
sulfonic acid in
0.9% NaC1 solution adjusted to pH 6.2, 6.6, 7.0, 7.4, and 7.8.
The cell reaction was quenched after 2 hr of cell incubation.
National
JV―-pentaceticacid; HEPES, N-2-hydroxyethylpiperazine
N'-2-ethane
3The abbreviations used are: DTPA, diethylenetriamine-N,N,N',N―, sulfonic acid; NTA, nitriotriacetic acid.
NOVEMBER
1974
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1974 American Association for Cancer Research.
2957
J. D. G!ickson et a!.
60
5
40
,
.
,
I
I
BICARBONATE
20
0
0
z
0
-20
0
PHOSPHATE
20
0
0
@
o-o@!
—0@0
0@—
°
0
-20
(I)
w
2
LACTATE
20
-J
z
0
x
0
2-20
I.—
I
@2O
I
z
TRIS
______,__@_____,
0
0
I—-20
z
w
20
0
.
MOPS0
.-000
@00.
TIME (hr)
@-2O
Chart 1. Time course of uptake of 6 ‘7
Ga citrate and 67Ga nitrate
20
by 7.0 X 106 L1210 leukemic cells,at pH 6.8 ±0.2 and 37°
.@
HEPES.0.000
0
-20
RESULTS
.0o..
-40
Chart 1 summarized the kinetics of in vitro binding to
L1210 leukemic cells of 2 commercial preparations of
carrier-free
67Ga, i.e., the citrate
concentration
proximate
@
@
@
and nitrate
of citrate
and nitrate
ions in the in
vitro incubation mixture were 1.6 X 10
M and 1.2 X 10@@
M, respectively. Zinc, formed by radioactive decay of 6 7Ga,
was estimated at a level of about 6.65 X 10
M. It has
previously been demonstrated that zinc at this concentration
does not influence the extent of in vitro 6 7Ga binding by
L1210 leukemic cells (8). Incubation at 37°was limited to 2
hi, during
which
time the exclusion
of the vital dye trypan
blue suggests retention of membrane integrity of the L1210
cells (8). During this time interval, the cells responded in an
identical
manner
to the citrate and nitrate
salts of 6 7Ga (Chart
1).
Chart 2 shows inhibition curves for nitrate, HEPES,
morpholinopropane sulfonic acid, Tris, lactate, phosphate, and
bicarbonate. Nitrate, HEPES, and morpholinopropane sulfonic
acid have no effect on in vitro 6 7Ga binding up to a
concentration of 102 M. Tris, lactate, and phosphate inhibit
@
0
salts. The 67Ga
-20
was 1.93 X 10-i ‘M for both samples. Ap
concentrations
slightly at concentrations
inhibits somewhat more.
above 10
M , whereas bicarbonate
Citrate, NTA, and EDTA are much
NITRATE.0
20
II
I0
-7
-6
?i
-5
-4
-3
-2
log [ANIoN]
Chart 2. Inhibition of 2-hr in vitro uptake of 67Ga by 7.0 x 106
L1210 leukemic cells incubated with sodium nitrate or various buffers
at pH 6.8 ±0.2 and 37°
. Each point, average of duplicate
measurements. The percentage inhibition was (C —C0)/C0 x 100%,
where C is the number of cpm measured for Li 210 cells incubated for
2 hr with the specified buffer and C0 is the number of cpm measured
for a control sample incubated with
Ga containing no citrate
other than that present in the commercial
sample. A typical experiment
measuring C0 appears in Chart 1.@MOPS, morpholinopropane sulfonic
acid.
The inert buffer morpholinopropane sulfonic acid (10 2M)
was used to measure cellular 67Ga uptake between pH 6.2 and
7.8 (Chart 4). A monotonic
the
radioisotope
incubation
decrease in cellular localization
is observed
with
increasing
of
pH of the
medium.
DISCUSSION
more potent inhibitors (Chart 3). Since the pH remained at 6.8
± 0.2
@
@
after
the
2-hr
incubation,
from the specific buffers
than from pH changes.
capacity is EDTA > NTA
phosphate, lactate, Tris >
HEPES, nitrate
0.
4 Concentrations
of
gallium
the
inhibitory
effects
The aqueous chemistry of gallium (III) is complex and not
originate
used in these experiments, rather
The relative order of inhibitory
> > citrate > > > bicarbonate >
morpholinopropane
sulfonic acid,
solution to yield Ga(H2O)6@'3 ions. However, the solubility
product
of Ga(OH)3
(about
5 X 10
7) (2) indicates
that the
maximum concentration of hydrated gallium (III) in neutral
solution is about 5 X 10-i 6M. Consequently, gallium (III) is
present in solution in the form of various complexes. The
(III)
have
been
calculated
from
the
i@Ci
of 67Ga measured, assuming the radiochemical was carrier free ; 1.05
@iCi
(2.60 x 108 mmoles) of 67Ga were administered.
2958
completely characterized
(2, 15). Salts such as the nitrate (18)
and perchlorate (5, 6) dissociate completely in strongly acidic
perchiorate
salt forms
metastable
polymers
with
maximum
degrees of polymerization near pH 3 (3, 14). Citrate polymers
have also been detected in this laboratory by equilibrium
CANCER RESEARCH VOL. 34-
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1974 American Association for Cancer Research.
Buffer andpHEffects on 67GaBinding
favor species with a greater or lesser permeability to tumor cell
I00
z 90
membranes
or with a greater or lesser affinity for cellular
receptors. The incorporation
of buffers, particularly citrate, in
commercial
preparations
of 6 7 Ga and the presence in the
280
plasma of various potential complexing agents of both low and
high molecular weight indicate a need to in'@stigate the effect
of counterions on tumor localization of this radioisotope.
I—
@60
@50
Inhibition
@40
@30
0:: 20
w
a.
6 7Ga
binding
by
Ll2lO
leukemic
cells
stable than intracellular
receptor complexes of the metal. This
theory is supported by the relative inhibitory capacity of the
buffers, which is in the order anticipated for the stability of
gallium (III) complexes, the chelating agents EDTA, NTA, and
0
0
-@7
-6
-5
-4
-3
citrate being the most potent inhibitors.
thermal analysis (7) and ion-exchange
-2
Moreover, differential
chromatography
(1)
demonstrate the formation of gallium (III) citrate complexes.
EDTA complexes with gallium (III) are well known from
Log [Buffer] M
Chart 3. Inhibition of 2-hr in vitro uptake of 67Ga by 7.0 x 106
L1210 leukemic cells incubated with sodium citrate (o), NTA (o), or
EDTA (V) at pH 6.8 ±0.2 at 37°.Each point, average of duplicate
measurements.4
titrimetric
studies
(13).
morpholinopropane
The
general
inert
response
of
sulfonic acid and HEPES to polyvalent
cations is also well-documented
(9).
a
z
The shapes of the EDTA, NTA, and citrate inhibition curves
(Chart 3) can readily be understood in terms of complex
formation. These curves resemble citration curves for metal
complex formation. At high buffer concentrations, all the
available metal may be sequestered in complexes unavailable
for tumor localization. These complexes, however, would
dissociate at low buffer concentrations by a mass law effect,
0
thereby
liberating
L12l0
cells may serve as a measure of the concentration
0
6 7Ga for tumor binding.
Binding
6 7Ga to
of
gallium (III) unassociated with buffer anions.
Taken together, these observations
suggest that the domi
nant cause for inhibition of in vitro 6 7Ga binding by various
(9
(I)
w
buffers is the competitive formation of gallium (III) complexes
with these buffers. However, the possibility that buffers may
interact in some manner with the cell to render it impermeable
-J
0
‘C
to gallium (III) or to block receptor
0
sites for this metal cannot
be totally discounted. Buffers such as citrate, phosphate, or
bicarbonate may also affect cellular metabolism.
The inhibitory effect of the buffers, at least that of the
chelating agents, does not result solely from competition for
cell surface-bound 67Ga. In the absence of buffer, only about
pH
Chart 4. The pH dependence of 67Ga bound by 7.0 x 106 L1210
leukemic cells incubated in vitro for 2 hr with 10 2 M morpholinopro
pane sulfonic acid at 37°
.@
dialysis (unpublished data). By analogy with other metal ions,
such as iron (III) (16, 17), which also undergo hydrolytic
polymerization, one anticipates that the polymeric equilibrium
probably shifts toward the formation of lower-molecular
weight complexes when buffer anions are present in great
excess (the condition under which the present study was
performed).
In addition,
various oxides of gallium (III) may be
present in neutral aqueous solution (15). Consequently, a
number of potential binders to tumor cells are present at
neutral pH. Changes in concentrations of counterions and
changes
6 7Ga
of
probably results from formation of 67Ga complexes that are
either impermeable to the tumor membrane or that are more
in pH may influence
to Li 210
NOVEMBER
cells
the extent
by shifting
of in vitro binding of
the molecular
equilibrium
to
12% of the cell-bound 6 7Ga appears to be adsorbed on the
cellular membrane
(8), whereas the inhibitory
capacities of
EDTA, NTA, and citrate approach 100%.
Inhibitory
effects
such as those described
in these in vitro
experiments may be significant in the design and interpreta
tion of in vivo studies of 6 7Ga localization. Average normal
concentrations of citrate in serum are about 7.5 X i0@ M
(19). This citrate concentration isjust below the level at which
significant
inhibition
of in vitro 6 7Ga uptake
by Li 21 0 cells is
observed (Chart 3). The observation by Konikowski et a!. (10)
that under in vivo conditions
citrate,
lactate,
and chloride
salts
of 67Ga localize to similar extents in tumors and are
cleared at similar rates is consistent with the conclusion that
citrate exerts a negligible inhibitory effect. However, these
authors did observe a significant diminution in the extent of
tumor localization of 67Ga when the DTPA salt was injected.
Spectrophotometric
constant
.
titration
studies indicate
.
for gallium (III) DTPA is about 10
that the stability
—28
°
at 20 (12).
1974
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1974 American Association for Cancer Research.
2959
J. D. Glickson et al.
Such stable complexes may indeed inhibit tumor localization
of 67Ga.
The effect of pH was studied
molecular
mechanism
of cellular
in order to explore the
6 7Ga incorporation.
In the
limited pH range used in these studies, tumor uptake of the
radioisotope
increased dramatically with decreasing pH.
Increasing uptake of the radioisotope in acidic media could be
caused by : (a) stabilization of molecular states of 67Ga
favorable for incorporation in the tumor, (b) titration of
cellular receptors, or (c) decomposition of cellular membranes
in acidic media. A definitive
choice between
these mechanisms
requires detailed information about the structure of the tumor
cell and the identification of the molecular species of gallium
(III) localized within tumor cells. If it is assumed that the first
5. Fiat, D., and Connick, R. E. Magnetic Resonance Studies of Ion
Solvation. The Coordination Number of Gallium (III) Ions in
Aqueous Solutions. J. Am. Chem. Soc., 88: 4754, 1966.
6. Fiat, D., and Connick, R. E. Oxygen-17 Magnetic Resonance
Studies of Ion Solvation. The Hydration of Aluminum (III) and
Gallium (III) Ions. J. Am. Chem. Soc., 90: 608—615,1968.
7. Gallet, J. P.. and Paris, R. A. Etude Thermometrique
Gallium. Anal. Clam. Acts, 39: 341—348,1967.
8. Glickson, J. D., Ryel, R. B., Bordenca, M. M., Kim, K. H., and
Gams, R. A. in vitro Binding of 67Ga to L1210 Cells. Cancer Res.,
33:2706—2713,
1973.
9. Good, N. E., Winget, G. D., Winter,W., Connolly, T. W., Izawa, S.,
effect,variations
in theconcentration
of tumorpermeable10.
species of gallium (III), contributes, at least in part, to the
observed variations of cellular uptake, the effect of pH could
be used
to help
identify
the species
of this element
required
to identify
11.
which
localize in Ll2lO cells. These would have to be species whose
concentration increases significantly between pH 8 and 6.
Additional data on the aqueous chemistry of gallium (III) is
12.
these species.
13.
ACKNOWLEDGMENTS
The authors are indebted to Dr. Paul Saltman, Dr. S. Y. Tyree, and
Dr. Phillip Aisen for helpful discussion of the data, to Dr. K. H. Kim
and Mary Sabens for technical assistance, and to Dr. Frank M. Schabel
and Mary W. Trader for the generous provision of L1210 leukemic
BD2F, mice.
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CANCER RESEARCH
VOL. 34
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Effects of Buffers and pH on in Vitro Binding of 67Ga by L1210
Leukemic Cells
Jerry D. Glickson, John Webb and Richard A. Gams
Cancer Res 1974;34:2957-2960.
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