[CANCER RESEARCH 35, 2985 2990, November 1975]
Tissue-specific Synthesis of Methotrexate Polyglutamates
in the Rat
V. Michael W h i t e h e a d , 2 M a r l e n e M. Perrault, and Susan Stelcner
Research Institute, Montreal General Hospital, Montreal, Quebec, Canada
SUMMARY
Purified tritium-labeled methotrexate was administered
to rats and Sephadex G-15 gel chromatography was used to
study the formation of poly-3,-glutamyl metabolites of
methotrexate in different tissues. These metabolites were
recognized as tritium-labeled antifolate fractions and were
identified by their response to serum pteroylpolyglutamate
hydrolases and by cochromatography with authentic standards. After doses equivalent to 15 to 20 mg for man, rat
liver and kidney were found to contain both 4-amino-10methylpteroylglutamyl-3,-glutamic acid and 4-amino-10methylpteroylglutamyl-3,-glutamyl-7-glutamic acid. With
one-third of the dose, only 4-amino- 10-methylpteroylglutamyl-3,-glutamic acid was found. Synthesis of methotrexate
polyglutamates in liver and kidney was limited to the
interval immediately following methotrexate administration
and appeared to occur by sequential addition of single
glutamyl residues to so-called "free" intracellular methotrexate. No synthesis of methotrexate polyglutamates was
demonstrated in small intestine or thymus. After formation,
4-amino-10-methylpteroyglutamyl-7-glutamic acid disappeared from liver and kidney with a half-time of 6.5 days.
The effect of these metabolites of methotrexate on cell
metabolism is unknown.
INTRODUCTION
M T X 3 (N-{p- {[(2,4-diamino-6-pteridinyl)methyl]methylamino}benzoyl}glutamic acid), an analog of the vitamin
folic acid (pteroylglutamic acid) and a potent inhibitor of its
metabolism, continues to be regarded as a potent and
effective antineoplastic agent after more than 2 decades of
use (6). Beneficial results have attended its use in the
treatment of a wide variety of neoplastic diseases. Strikingly, its use in the treatment of acute lymphoblastic
leukemia of childhood (1 1) and of choriocarcinoma (16),
usually in combination with other agents, has resulted in
cures. In addition, it has proved of value in the treatment of
psoriasis (19) and as an immunosuppressive agent (18).
As with other antineoplastic agents, the effectiveness of
MTX has been limited by its toxicity toward normal tissues,
particularly bone marrow and gastrointestinal mucosa, and
by the inherent or acquired resistance of neoplastic tissues
to its cytotoxic action. While the subsequent administration
of folinic acid (Dc-5-formyltetrahydrofolate) has been found
to reduce host toxicity (8), permitting the use of exceedingly
large doses of MTX (5), the problem of resistance remains
unsolved. This has been thought to be due to one or another
of 3 different mechanisms. (a) Resistance has been associated with a reduction in the permeability or active membrane transport of MTX into cells (7). (b) MTX administration has been found to result in the "induction" of synthesis
of the target enzyme dihydrofolate reductase in human
leukocytes and erythrocytes (3, 10). (c) Certain tissues have
the capacity to metabolize MTX. Thus, oxidation of MTX
is extensively carried out in rabbit liver and, to a lesser
extent, in guinea pig liver but has not been detected in the
livers of other species, including man (12). Again, certain
bacteria, including those present in mouse intestine, contain
an enzyme capable of hydrolyzing MTX to glutamic acid
and 4-deoxy-4-amino- 10-methylpteroic acid (14).
Recently, an additional mode of metabolism of MTX was
discovered. Nair and Baugh (15) synthesized a series of
poly-7-glutamyl metabolites of MTX and with their aid
were able to identify small amounts of MTX(G1) and
MTX(G2) in rat tissues (2). Evidence of formation of similar
metabolites of MTX in mouse liver was also reported (21).
The present communication provides confirmation of the
synthesis of MTX(G1) and MTX(G2) in rat liver and kidney
and presents evidence that such synthesis is dose dependent,
can be significant in amount, and is specific to certain
tissues. Preliminary reports of some of these findings have
been presented (21, 22).
MATERIALS AND METHODS
Groups of 3 to 5 male adult Sprague-Dawley rats were
1This work was supported by Grant MA-2384 from the Medical housed in wire-bottomed metabolic cages and fed a comResearch Council of Canada and by a grant from the Cancer Research
merical diet (laboratory chow; Ralston Purina Co. of
Society, Inc.
2To whom reprint requests should be addressed, at Division of Canada Ltd., Woodstock, Ontario, Canada) and water a d
Haematology, Montreal General Hospital, 1650Cedar Avenue, Montreal, l i b i t u m . [3H]MTX was usually administered s.c. in l, 3, or
Quebec, Canada.
9 doses. At intervals after the final dose, animals w e r e
3The abbreviations used are: MTX, methotrexate; MTX(G0, methotrexate monoglutamate; MTX(G2) methotrexate diglutamate; [3H]MTX, anesthetized with ether, blood was withdrawn from a
jugular vein, and organs were removed. Liver, kidney, and
[Y,5'-9(n)-3H]methotrexate.
Received May 31, 1974; accepted July 15, 1975.
thymus were rapidly weighed and homogenized in cold 100
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2985
V. M . W h i t e h e a d et al.
mM sodium phosphate buffer containing ascorbic acid, 150
mg/100 ml, final pH 7.0. The small intestine was rinsed
with 0.9% NaC1 solution and blotted dry, and the proximal
one-third was weighed and homogenized as described above.
Homogenates were heated for 5 rain in boiling water and
centrifuged, and the supernatants were stored. The preparation and heating of extracts were completed in less than 20
min, since studies revealed that hydrolysis of pteroylpolyglutamates in unheated rat liver extracts did not commence
within this interval of time (unpublished data). Blood
samples were mixed with an equal volume of 0.5 y
perchloric acid (Allied Chemical Co., Morristown, N. J.)
and centrifuged, and the supernatants were stored.
Extracts were initially stored at 4 ~ to avoid possible
hydrolysis of poly-7-glutamyl metabolites (4), but these
proved unstable. Thereafter, extracts were stored at - 2 0 ~,
following the demonstration that hydrolysis did not occur
with freezing. The 3H in extracts and in chromatographic
fractions was counted in a Packard Tri-Carb Model 2425
liquid scintillation spectrometer using a commercial scintillant (Aquasol; New England Nuclear, Boston, Mass.).
Results were expressed as ng M T X or M T X equivalent per
g tissue or ml blood.
Gel chromatography was carried out using Sephadex
G-15 gel (Pharmacia Co., Uppsala, Sweden) and 0.9- x
55-cm columns (20). The gel was swelled in boiling water for
1 hr and columns were poured by gravity. Columns were
eluted at room temperature with 25 mM sodium phosphate
buffer containing 200 mM mercaptoethanol (Eastman
Kodak Co., Rochester, N. Y), final pH 7.0, at a flow rate of
20 to 25 m l / h r , collecting 1.7- to 2.0-ml fractions. Later,
100 mM NaC1 were substituted for mercaptoethanoi without
altering the elution of M T X or its metabolites. Void and
total column volumes were determined with hemoglobin
and tritiated water and usually corresponded to Tubes 7 and
16, respectively.
Folate was assayed using Lactobaeillus casei (American
Type Cell Culture 7469) (1, 23). To provide a greater
contrast in chromatograms between regions of growth of L.
casei and regions of inhibition of growth, due to the
presence of M T X and its metabolite(s), folic acid, 0.5 ng,
was added to all assay tubes.
Hydrolysis of poly-7-glutamyl metabolites of M T X in
liver extracts was carried out using fresh human serum as a
source of pteroylpolyglutamate hydrolase(s) as previously
described for the hydrolysis of pteroylpolyglutamates in
human liver (20). Equal volumes of fresh human serum and
of pooled chromatographic fractions were mixed and incubated for 90 min at 37 ~ With such treatment, complete
hydrolysis of tritium-labeled liver pteroylpolyglutamates to
oligoglutamates and monoglutamates has been demonstrated
when the pH was 4.6, while no hydrolysis occurred when
incubation was carried out at pH 7.0 (unpublished data).
[3H]MTX was obtained as the sodium salt from Amers h a m / S e a r l e Co., Don Mills, Ontario, Canada, and was
mixed with commercial M T X (Lederle Products Department, C y a n a m i d of Canada Ltd., Montreal, Quebec, Canada) to obtain a specific activity of 1 or 2 /zCi/ug. This
mixture was purified by gel chromatography prior to use
(Chart 1). p-Aminobenzoyl-L-glutamic acid was obtained
2986
from Nutritional. Biochemical Corp., Cleveland, Ohio.
MTX(G1) and MTX(G2) were obtained as gifts from Dr.
Nair and Dr. Baugh (15). 4-Deoxy-4-amino-10-methylpteroic acid was a gift from Dr. Y. S. Shin (17). The
chromatographic behavior of standard materials was determined spectrophotometrically in a Beckman DK2 recording
spectrophotometer.
RESULTS
Sephadex G-15 Gel Chromatography of MTX
Chart 1 shows chromatograms of [aH]MTX before and
after purification. M T X bound tightly to the gel, appearing
in about 2 column volumes. The contaminant in Tube 12
(Chart IA) cochromatographed with para-aminobenzoyI-Lglutamic acid. The identity of other impurities was not
determined. 4-Deoxy-4-amino-10-methylpteroic acid eluted
in Tube 88 and appeared to be a contaminant of unlabeled
M T X but not of [aH]MTX. A number of contaminants of
unlabeled M T X was detected spectrophotometrically. None
appeared to correspond to contaminants present in
[3H]MTX. When unpurified [aH]MTX was administered to
rats, chromatograms of liver, kidney, and thymus revealed
the presence of materials corresponding to 2 contaminant
fractions (Chart 1A, Tubes 15 and 44) in these tissues. This
was particularly striking when tissues were examined several days after injection. These contaminants were not
detected following administration of purified [3H]MTX
(Chart IB). Purified [3H]MTX was stored at 4 ~ and
remained pure for as long as 2 months under these
conditions (Chart I B). At the same time, there appeared to
be no exchange of the tritium label with the aqueous storage medium.
Identification of MTX(G1) in Rat Liver and Kidney
A typical Sephadex G-15 gel chromatogram of a heated
extract of rat liver is shown in Chart 2. This extract was
prepared from a 556-g rat 24 hr after the last of 5 daily i.p.
injections of 20 ug [aH]MTX. Two major tritium-labeled
fractions were present: MTX (Tube 43) and an unknown
material (Tube 21). Both fractions were inhibitory to growth
of L. casei. A similar chromatogram was obtained from an
extract of kidney from the same animal.
When folic acid was added to assay tubes containing
inhibitory quantities of the unknown material, growth of L.
casei was restored, demonstrating that this tritium-labeled
material was a folate antagonist. To determine whether this
material was a poly-7-glutamyl metabolite of MTX, aliquots of the material were incubated with fresh human
serum [as a source of pteroylpolyglutamate hydrolase(s)]
for 90 min at 37 ~ The mixture was then heated to coagulate
protein and the centrifuged supernatant was rechromatographed. No change in the Sephadex G-15 chromatographic
behavior of the unknown material occurred when incubation
was carried out at pH 7. However, after incubation at pH
4.6, the unknown material was found to chromatograph in
the position of M T X . These results demonstrated that the
CANCER RESEARCH VOL. 35
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Research.
MTX
Polyglutamates
dpm/tube x 10 5
4
~t
dpm/tube x I06
3
A 304 nm
08
ii.
~
fi
',
I
B
'
0
8
16-
24
32
40
tube number
j
48
56
O'
8
16
24
32
tube number
1
40
48
Chart 1. Sephadex G-15 gel chromatograms of [3H]MTX before and after purification. A, cochromatography demonstrated that MTX(G1) (- - -)
and MTX(G2) (. . . . ) were not contaminants of [3H]MTX. See text for further details. B, chromatogram of purified [3H]MTX after storage at 4~ for
30 days. An identical chromatogram was obtained after 60 days. Vo, void volume.
unknown material was a poly-7-glutamyl metabolite of
MTX and not a degradation product. The identity of this
material was established by cochromatography with authentic standards. As shown in Chart 3, the unknown material in
extracts of rat liver and kidney appeared after MTX(G2)
and cochromatographed with MTX(G1).
Synthesis of MTX Polyglutamates in Rat Tissues. Table 1
contains results of 2 studies carried out to determine the
effect of time after injection of [3H]MTX on the synthesis of
MTX polyglutamates in rat liver. No increase in concentration of MTX(G1) appeared to occur in the interval between
the earliest time studied (1 and 4 hr postinjection, respectively) and 24 hr. These results indicated that synthesis of
MTX(Gx) had essentially ceased by the time initial samples
were studied, being limited to an interval of time immediately following injection. At the same time, these results
demonstrated that the time of examination of tissues
following injection was not a critical factor in assessing
differences of MTX polyglutamate synthesis.
In a series of experiments, the concentration of MTX(G1)
in liver was measured after administration of [3H]MTX at a
dose of 80 ug/kg. With this dose, the concentration of
MTX(G1) was greater with 3 divided doses than with a
single dose and was greater after parenteral than after p.o.
administration. These observations were consistent with .the
hypothesis that synthesis of M T X polyglutamates was
limited to intervals during which the extracellular concentration of M T X was high. Maximum concentrations of
M T X polyglutamates for a given dose of [3H]MTX were
found with s.c. administration in divided doses.
Synthesis of M T X polyglutamates was dose dependent
and tissue specific. At a dose of 80 #g/kg, no synthesis of
M T X ( G 0 was detected in either small intestine or thymus
(Chart 4 and Table 2). In contrast, this dose resulted in
significant concentrations of MTX(G1) in both liver and
kidney (Table 2). A 3-fold increase in the dose of [3H]MTX
yielded a higher concentration of M T X ( G 0 in liver, and this
ng/tube/g
liver
A 6 4 0 nm
25--
1.00 -
20 --
0.80
15
0.60
]0
0.40
5
0.20
0
0.00
0
.............
8
16
24
tube
32
40
48
56
number
Chart 2. Sephadex G-IS gel chromatogram of an extract of rat liver
prepared after administration of [3H]MTX. Folate in fractions was assayed
with L. casei (O) and 3H was counted (O). See text for details.
was accompanied by the appearance of an additional
tritium-labeled fraction. This material was inhibitory to
growth of L. casei and was identified as MTX(G2) by
cochromatography with authentic standards (Chart 5). At
the highest doses tested, both rat liver and kidney were
found to contain MTX(G1) and MTX(G2) in significant
amounts. In contrast, synthesis of MTX polyglutamates in
rat small intestine did not occur, except possibly in trace
amounts following the highest dose used (Table 2).
Fate of MTX(G1) in Rat Liver and Kidney
To determine whether M T X ( G 0 persisted in liver and
kidney, groups of rats received 4 daily s.c. injections of
[3H]MTX, 20 tsg/kg, and were sacrificed 1, 4, 7, and 11
days after the last dose. Initial chromatograms revealed that
MTX(G1) comprised 49.3 and 19.6% of the 3H in pooled
extracts of liver and kidney, respectively. Succeeding chromatograms of extracts revealed that MTX(G1)disappeared
exponentially at a similar rate from both organs (Chart 6).
No MTX(G2) was formed during this interval of time. In a
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2987
V. M .
W h i t e h e a d et al.
2nd study, rats received a single injection of [ S H ] M T X , 80
# g / k g , and e x t r a c t s of t h e i r livers a n d k i d n e y s were p o o l e d
and c h r o m a t o g r a p h e d 26 d a y s l a t e r ( T a b l e 1). D u r i n g this
interval, t h e r e was a 23.4% fall in t o t a l liver M T X f r o m 1.88
+ 0.30 to 1.44 • 0.12 #g ( m e a n + S.D.). A f t e r 26 days, 92%
and 96% of the r e m a i n i n g 3H in liver a n d k i d n e y , respectively, c h r o m a t o g r a p h e d as M T X . M T X ( G 1 ) was not
identified in these c h r o m a t o g r a m s . T h e s e results d e m o n s t r a t e d t h a t M T X ( G 1 ) did not persist in rat tissues in
c o n t r a s t to M T X itself. N o r was it c o n v e r t e d to M T X ( G = )
with time.
DISCUSSION
T h e a p p a r e n t p r e f e r e n t i a l a n d progressive a c c u m u l a t i o n
in rat tissues of 2 t r i t i u m - l a b e l e d c o n t a m i n a n t m a t e r i a l s
p r e s e n t in [ 3 H ] M T X r e n d e r e d m a n d a t o r y the p u r i f i c a t i o n of
[ 3 H ] M T X p r i o r to use. T h i s was easily achieved by gel
c h r o m a t o g r a p h y , p o o l i n g the large [ 3 H ] M T X p e a k but
d i s c a r d i n g the final tubes of the p e a k t h a t c o n t a i n e d 1 of the
o f f e n d i n g c o n t a m i n a n t s . T h e p r o d u c t r e m a i n e d stable for as
long as 2 m o n t h s in a q u e o u s m e d i u m at 4 ~ and was
c a l c u l a t e d to be 99.7% pure w h e n r e c h r o m a t o g r a p h e d on a
ng/g
32
28
24
24
20
20
16
A
302 nm
1.00
16
i'i
12
8
).80
iver
kidney
12
i"
9" i
t
O.6O
)
8
I
0.40
-0.20
4
0
0
,-----~--g~
8
16
24
32
40
--.~
0 .
.
48
60
0
8
tube number
.
.
16
.
24
.
.
32
40
-0.00
48
Chart 3. Cochromatography of extracts of rat liver and kidney with M T X ( G ~ ) ( - - - )
60
and M T X ( G = ) ( . . . . ).
Table 1
The effect of time after injection on synthesis of M T X ( G l) in rat liver
Rats were given s.c. injections of [3H]MTX, 80 #g/kg, and liver extracts were prepared at
intervals thereafter. Pools of extracts from each time interval were chromatographed on Sephadex
G-15. Results are expressed as ng MTX or ng equivalents of MTX per g liver.
Study
No. of
rats
MTX(G1)
(ng/g)
MTX
(ng/g)
Total
(ng/g)
4 hr
8 hr
26 hr
3
3
4
73
65
83
143
148
150
219
214
236
1 hr
24 hr
26a days
3
3
4
33
35
0
216
229
98
250
265
107
Time
Total
(ng/liver)
Blood
(ng/ml)
4.1
3.3
4.2
1827
1942
1440
8.9
4.5
0.0
a Rats killed at 26 days were part of Study B.
A
302 nm
ng/g
16
20
I
thymus
^
~6 t
[',
12
1.00
I
12
G.I. tract
" 'I~'
,"~
0.80
)1
-0.60
8
4
0
I...._ _ ; ; / % _ = .
0
8
16
24
32
40
48
4
).20
0
60
0
--:--=. . . . . . . . . . . .
8
16
-'------,-"
24
32
40
48
--/J*-- ~ ).00
60
tube number
Chart 4. Cochromatography of extracts of thymus and small intestine with MTX(G 1) (-
2988
) and MTX(G2) ( . . . . ). G.I., gastrointestinal.
C A N C E R R E S E A R C H VOL. 35
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MTX
n u m b e r of occasions. Even with this purity, however, a
problem could still be encountered. Thus, in a recent study,
a rat received 75 mg of M T X c o n t a i n i n g 600 #Ci of purified
[3H]MTX in a 6-hr infusion. G r e a t e r than 90% of the
tritium in liver, kidney, small intestine, and brain c h r o m a t o graphed as a c o n t a m i n a n t ( C h a r t 1,4, Tube 15) and
appeared to originate from the l - # C i a m o u n t of this
m a t e r i a l present in the infusate. Should this material be
present as a c o n t a m i n a n t in unlabeled M T X , it is interesting
to speculate on its effect on tissues of patients treated with
very large doses of M T X (5).
These studies provided c o n f i r m a t i o n of the synthesis of
poly-3,-glutamyl metabolites of M T X in rat tissues (2). In
addition, the findings delineated a n u m b e r of specific
features concerning this m e t a b o l i s m . U n l i k e the synthesis of
p t e r o y l p o l y g l u t a m a t e s from folic acid in m a m m a l i a n tissues, in which successive g l u t a m y l residues are added to
folate to yield p r e d o m i n a n t l y p t e r o y l p e n t a g l u t a m a t e s and
p t e r o y l h e x a g l u t a m a t e s after 24 to 48 hr and, with further
time, some p t e r o y l h e p t a g l u t a m a t e s (13), no evidence of
progressive addition of g l u t a m y l residues to M T X with time
A
302 nm
ng/g
40 -
0.8
.,
',
ii
II
II
was d e m o n s t r a t e d . R a t h e r , synthesis of M T X polyglutamates appeared to be a p h e n o m e n o n of short d u r a t i o n ,
limited to the interval i m m e d i a t e l y following [~H]MTX
a d m i n i s t r a t i o n . The q u a n t i t y of M T X p o l y g l u t a m a t e s synthesized was clearly dose dependent. In addition, with the
same dose, a greater synthesis was seen with p a r e n t e r a l than
with p.o. a d m i n i s t r a t i o n and with the use of divided doses.
At the highest doses studied, there was f o r m a t i o n of both
M T X ( G 1 ) and MTX(G2) in rat liver and kidney, while at
lower doses only M T X ( G I ) was found. l h e s e observations
suggested that synthesis of M T X p o l y g l u t a m a t e s occurred
by sequential addition of single g l u t a m y l residues to socalled free intracellular M T X (9). It appeared that M T X
bound to d i h y d r o f o l a t e reductase was not a substrate for
M T X p o l y g l u t a m a t e synthesis. It was not clear why
MTX(G2) f o r m a t i o n occurred with large divided doses of
M T X but did not arise with time from MTX(G1) after this
had been formed in liver and kidney. Possibly, M T X ( G 0
b e c a m e bound within the cell soon after its f o r m a t i o n and,
in t h a t state, was no longer a substrate for addition of
further g l u t a m y l residues.
The persistence of M T X in tissues has been a t t r i b u t e d to
its pseudoirreversible binding to the e n z y m e d i h y d r o f o l a t e
reductase with stabilization of the complex (10). Such
persistence was d e m o n s t r a t e d in rat liver and kidney in the
!i ,,
ng/g
ii J'
32
ii
Polvglutamates
',
--.
100
0.6
J i;
it
24
0.4
T~/2 6.6 da;s ' - ~ - ~ - -
!
Liver
-
16
0.2
-
0.0
-
10
0
8
16
24
32
40
t.o
48
T~/2 6.5 days
--
-~,
Kidney
I
1
I
I
I
4
7
11
tube. number
days
Chart 5. Cochromatography of a liver extract with MTX(GD (
-)
and MTX(G2) ( . . . . ). The rat received [3H]MTX, 240 #g/kg, in 9 divided
s.c. doses at 15-min intervals and was killed 2 hr later (see Table 2).
Chart-6. Levels of MTX(G1) in pooled extracts of rat liver and kidney
at intervals following the last of 4 daily s.c. injections of [aH]MTX, 20
#g/kg. T~, half-time.
Table 2
Levels of MTX, MTX(G1), and MTX(G2) in rat tissues following s.c. [3H]MTX
The dose was divided and administered in 3 or 9 injections of about 27 #g/kg at 15- or 30-min intervals, and animals were killed 4 hr after the 1st dose.
Results are expressed as ng MTX or equivalent per g tissue.
Liver (ng/g)
Dose
No. of
rats
88
77
80
240
241
287
298
l
3
3
1
1
1
1
M T X ( G 2 ) MTX(G1)
0.0
0.0
2.5
37.0
44.2
!7.0
59.3
90.3
22.0
60.9
143.6
77.9
103.6
228.8
Kidney (rig/g)
Small intestine (ng/g)
Thymus (ng/g)
MTX
MTX(G~)
MTX(G0
MTX
MTX(G,)
MTX
MTX(G,)
MTX
155.6
233.7
190.4
70.6
102.8
139.8
142.9
0.0
0.0
0.0
3.2
3.7
19.9
18.1
30.2
21.5
20.0
28.2
22.0
59.6
58.5
216.0
216.7
178.8
173.1
179.9
314.3
291.4
0.0
0.0
0.0
0.0
0.0
0.0
2.0
73.8
61.0
49.5
73.4
128.7
368.5
150.7
0.0
0.0
0.0
50.9
29.1
37.3
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V. M .
Whitehead
e t al.
p r e s e n t studies. In c o n t r a s t , t h e r a t e o f d i s a p p e a r a n c e o f
M T X ( G D f r o m rat liver a n d k i d n e y a p p e a r e d to be a b o u t 8
t i m e s f a s t e r t h a n t h a t o f M T X . W h e t h e r this r e s u l t e d f r o m
loss o f a specific p o p u l a t i o n o f cells c o n t a i n i n g h i g h levels o f
M T X ( G D , from d e g r a d a t i o n a n d / o r release of M T X ( G ~ )
f r o m cells, or f r o m c o n v e r s i o n o f M T X ( G 1 ) to M T X w a s
n o t d e t e r m i n e d . T h u s , it w a s n o t c l e a r w h e t h e r t h e poly-"t'g l u t a m y l m e t a b o l i t e s o f M T X s e r v e d as an i n t r a c e l l u l a r
r e s e r v o i r o f M T X or not.
T h e a p p a r e n t i n a b i l i t y of s m a l l i n t e s t i n e a n d t h y m u s to
synthesize poly-,y-glutamyl metabolites of M T X m a y have
b e e n d u e to t h e i r f a i l u r e to a c c u m u l a t e high levels o f
i n t r a c e l l u l a r M T X . In s u p p o r t o f this w a s t he f i n d i n g o f
s m a l l a m o u n t s o f M T X ( G D in s m a l l i n t e s t i n e with t h e
h i g h e s t d o s e a d m i n i s t e r e d . H o w e v e r , s y n t h e s i s w a s n o t seen
w h e n M T X wa s fed with t h e i n t e n t o f e n s u r i n g e x p o s u r e o f
t h e s m a l l i n t e s t i n e to h i g h levels of t he d r u g . W h a t e v e r be
t h e m e c h a n i s m , t h e s e d i f f e r e n c e s in a bi l i t y to s y n t h e s ! z e
M T X p o l y g l u t a m a t e s w e r e seen w i t h d o s e s of M T X w i t h i n
t h e u s u a l t h e r a p e u t i c r a n g e used in m a n . S i n c e s m a l l
i n t e s t i n e a n d , p r e s u m a b l y , t h y m u s (18) d i f f e r m a r k e d l y
f r o m liver a n d k i d n e y in t h e i r s u s c e p t i b i l i t y to M T X
i n t o x i c a t i o n , it m i g h t be s p e c u l a t e d t h a t s y n t h e s i s of M T X
p o l y g l u t a m a t e s serves to p r o t e c t cells f r o m t h e l e t h a l effect
o f this d r u g . H o w e v e r , at t h e p r e s e n t t i m e , t he effect o f t h e s e
m e t a b o l i t e s o f M T X on cell m e t a b o l i s m is u n k n o w n .
ACKNOWLEDGMENTS
The authors thank Grete Bridgewater and Maureen McCormack for
technical assistance and Nancy George for aid in preparing this manuscript.
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C A N C E R R E S E A R C H VOL. 35
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1975 American Association for Cancer
Research.
Tissue-specific Synthesis of Methotrexate Polyglutamates in
the Rat
V. Michael Whitehead, Marlene M. Perrault and Susan Stelcner
Cancer Res 1975;35:2985-2990.
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