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Transport of L-cystine and L-djenkolic acid by the in vitro small intestine
Vol . 5, pp . 1163-116, 1966 . Life Sciences Printed in Great Britain .
Pergamon Press Ltd.
TRANSPORT OF L-CYSTINE AND L-DJENKOLIC ACID BY THE IN VITRO SMALL INTESTINE Richard P . Spencer, Kenneth R. Brody, Francis Viahno Section of Nuclear Medicine, Department of Radiology, Yale University School of Medicine, New Haven, Connecticut
(Received 4 March 1966 ; in final form 5 May 1966) Reports that intestinal absorption of cysteine was unimpaired is cystinuric subjects, while that of cyetine was defectivel, suggested a role for the disulfide bond in the normal transport event.
One approach to elucidating
thin possible participation was to employ analogues in which -S-S- was replaced by other moieties (such as -S-CH2-S- in djeakolic acid, and -CH2 CH2- in diaminopimelic acid) .
Lin and co-workera 2 reported that djenkolic acid was not traae-
ported against a concentration gradient by the hamster small intestine is vitro , and Randall and Evered 3 described lack of transport of diaminopimelic acid by rat intestinal sacs .
Such observations were extended by this laboratory when
additional analogues were tested and found to be without transport against a concentration gradient by hamster intestinal sacs 4 .
However,
since E . coli
has been stated to have a transport system for diamiaopimelic acid s, it was deemed essential to pursue these studies in an attempt to optimize airy minimal transport of L-djeakolic acid by the mammalian small intestine . Methods Adult golden hamsters and C3H mice were obtained from commercial auppliers .
Unlike the procedure previously utilized, they were fasted for 16
hours prior to use .
Following sacrifice,
the small intestine was averted and
Supported by Grants CA 06519, AM 09429 and AM 07958 from the U.S . Public Health Service
1163
1164
INTESTINAL TRANSPORT
washed in pH 7.4 Rrebe-bicarbonate buffer . buffer .
Yol . 5, No . 13
No glucose was present in the
The hamster small intestine was divided into three sacs in some cases
(each filled with one ml .) or into six sacs (each filled with 0 .5 ml .) . intestinal sacs
(three per animal) were filled with one ml . of fluid.
Mouse Sacs
were placed in five ml . of fluid of identical composition (the compound dissolved in buffer),
gassed with 95~ 02 + 5% C02 , and incubated in a shaking water bath
at 37 °C for one hour .
At the end of incubation, sacs were drained into gradu-
ated centrifuge tubes (to measure any volume change) and weighed.
Mucosal and
eerosal . solutions were centrifuged to remove sloughed tissue, and asesyed by liquid scintillation counting .
Since the initial condition was the same con-
centration of compound inside and outside of the intestinal eaca, a~ increase in the inside (serosal) concentration in the absence of water lose represented movement against a concentration gradient .
The compounds utilized ware L-
cystine- 3H (Schwarz Bioresearch) and L-djenkolic acid- 3H prepared by a special technique that will ba described in detail in a separate cammunicatioa; the djeakolic acid was essentially entirely in the Inform, aé determined by optical rotation .
Carrier was added as necessary.
Periodic chromatographic checks of
the mucosal and serosal solutions revealed no radioactivity outside of the ra dioactive peak corresponding to Qie authentic compound .
Inhibition studies
were carried out by adding nonlabeled amino acids to the mucoeal and serosal fluids to determine if they interfered with L-djenkolic acid transport . Results In the absence of glucose, water transport by the intestinal eaca was is the mucoeal to aeroeal direction and was negligible
(0-0 .1 ml, gained) .
Hence as increase is radioactivity in the aeroeal fluid represented transport and not increased concentration due to water loan . djeakolic acid of 1 x lÔ
4
At a concentration of Ir
M, when three sate were made from the small intes-
tine of èach hamster, the maximal concentration observed was 1 .04 (aeroeal concastration/standard concentration) with a probable error of 0 .05 due to pipetting and counting .
This is consistent with the previous inability to demonstrate
INTESTINAL TRANSPORT
Vol . S, .No . 13
transport against a concentration gradient .
1165
In an effort to maximize any trans-
port, movement of L-cyetine was next studied as a function of the site of oriResults are shown in Fig. 1 .
gin of the gut sac .
More was transported is the
fifth of six sacs from the hamster small intestine ; approximately 2 .6 times more was transported in the fifth sac than in the first . Accordingly, L-djenkolic acid transport studies were repeated, using only the fifth sac out of six prepared from the hamster's small gut . 1 x lÔ
4
At
M, the ratio of aeroeal concentration/standard concentration was 1.12 .
This was the mean of 12 such sacs ; transport was demonstrable in ten.
40 q 30 ' 0
N
m .-1
20 '.
1
2
3
5
4
Sac Number FIG . 1 Serosal gain of L-cystine (using 1 x lÔ Bach point is the mean of six hamsters .
4
M) .
Recognizing that other species might more readily transport L-djenkolic acid than the hamster, the mouse gut, of 1 x
10
4
the mouse small intestine was also employed .
Using
transport of L-djenkolic acid was demonstrable at coacentrationa
M to 1 x
10
9
M in all sacs .
The maximum ratio of the aeroeal con-
centration/standard concentration was 1.21 at 1 x 10
4
M L-djenkolic acid .
Over the small concentration range open to study (limited at the higher range by insolubility, and at the lower range by the aecesaity for adequate atatiatics of counting),
fully adequate kinetic investigation could not be obtained .
The maximum transport of L-djenkolic acid by the mouse small Lateatiae vas
INTESTINAL TRANSPORT
1166 approximately 0 .3
Vol . 5, No . 13
moles/gm . tissue, while the apparent Michaelie coastant was
on the order of 2 x
10 4
M.
Due to the limited concentration range in which L-djenkolic acid could be utilized, definitive studies on inhibition of transport by other amino acids were not possible .
However,
hibited over 90X by 5 x
10
tion obtainable (under 1 x transport of 1 x lÔ
4
3
transport of 5 x lÔ M L-lysine .
10
3
4
M L-djenkolic acid was in
At the maximum L-cyatine concentra-
M), there was no significant depression of the
M L-djenkolic acid . Discussion
The spatial distribution of L-cyatine movement by the in vitro hamster small intestine is similar (when averaged over several animals) to that of the neutral amino acids, and the amino acida6. reported for the rats .
However,
it differs from that
Utilizing the conventional techniques of three sacs
per hamster, no clear transport of L-djenkolic acid could be demonatrated 4 . However,
by concentrating on the likely site of maximal movement, slight concen-
tration has been shown by the present experiments . analogues of cyatine, previously studied in the D,Lsacs per hamster, lined here .
They suggest that other form and by using three
should be repeated under the special transport conditions out-
That is, the hamster should be fasted, glucose should be absent
from the medium, and the fifth of six intestinal sacs per animal should be employed .
Transport of L-djenkolic acid by the mouse small intestine may serve
to emphasize the observation that this preparation maintains its viability following removal from the body, and gives transport kinetics that approach the in vivo situation$ . Summary The spatial distribution of L-cyatine transport by the in vitro hamster small intestine was described .
More was transported five-sixths of the way
dawn the gut than at other locales .
Using hamster intestinal sacs made from
this area only, in buffer devoid of glucose (to reduce water transport) and L-
Vol .
5, No . 13
djenkolic acid at 1 x
116
INTESTINAL TRANSPORT 10
4
M, miaimal transport against a concentratioa grad-
seat was demoastrable in about 90R of the experiments .
Utiliziag the mouse
small intestine, all sacs transported L-djenkolic acid against a conceatratioa gradient .
Transport was inhibited by L-lysine .
The other D,L- analogues of
cyatine previously noted not to be transported should be re-investigated employing the L-isomers and the conditions outlined here with the hamster gut,
or
preferably with the small intestine of the mouse . RefereACes 1.
L.B . Rosenberg, J.L . Durant and J .M . Holland, New ßiüi . J. Med . 273, (1965) .
1239
2.
S .C .C . Lin, H. Hagihira aad T .H . Wflson, Am . J . Phvaiol. 202, 919 (1962) .
3.
H.G . Randall and D .F . Svered, Biochim. Biouhus . Acta = , 98 (1964) .
4.
R.P . Spencer, K .R . Brody aad H.G . Mautaer, Nature 207, 418 (1965) .
5.
L. Leive aad B .D . Davis, J. Biol . Chem . 240, 4362 (1965) .
6.
R.P . Spencer, K .R . Brody and B.M . Lottere, Biochim. Bioohvs . Acta â8, 400 (1964) .
7.
M.W . Neil, Biochem. J . ~, 118 (1959) .
8.
W. Wright, Fed. Proc . ~, 416 (1966) .
Pergamon Press Ltd.
TRANSPORT OF L-CYSTINE AND L-DJENKOLIC ACID BY THE IN VITRO SMALL INTESTINE Richard P . Spencer, Kenneth R. Brody, Francis Viahno Section of Nuclear Medicine, Department of Radiology, Yale University School of Medicine, New Haven, Connecticut
(Received 4 March 1966 ; in final form 5 May 1966) Reports that intestinal absorption of cysteine was unimpaired is cystinuric subjects, while that of cyetine was defectivel, suggested a role for the disulfide bond in the normal transport event.
One approach to elucidating
thin possible participation was to employ analogues in which -S-S- was replaced by other moieties (such as -S-CH2-S- in djeakolic acid, and -CH2 CH2- in diaminopimelic acid) .
Lin and co-workera 2 reported that djenkolic acid was not traae-
ported against a concentration gradient by the hamster small intestine is vitro , and Randall and Evered 3 described lack of transport of diaminopimelic acid by rat intestinal sacs .
Such observations were extended by this laboratory when
additional analogues were tested and found to be without transport against a concentration gradient by hamster intestinal sacs 4 .
However,
since E . coli
has been stated to have a transport system for diamiaopimelic acid s, it was deemed essential to pursue these studies in an attempt to optimize airy minimal transport of L-djeakolic acid by the mammalian small intestine . Methods Adult golden hamsters and C3H mice were obtained from commercial auppliers .
Unlike the procedure previously utilized, they were fasted for 16
hours prior to use .
Following sacrifice,
the small intestine was averted and
Supported by Grants CA 06519, AM 09429 and AM 07958 from the U.S . Public Health Service
1163
1164
INTESTINAL TRANSPORT
washed in pH 7.4 Rrebe-bicarbonate buffer . buffer .
Yol . 5, No . 13
No glucose was present in the
The hamster small intestine was divided into three sacs in some cases
(each filled with one ml .) or into six sacs (each filled with 0 .5 ml .) . intestinal sacs
(three per animal) were filled with one ml . of fluid.
Mouse Sacs
were placed in five ml . of fluid of identical composition (the compound dissolved in buffer),
gassed with 95~ 02 + 5% C02 , and incubated in a shaking water bath
at 37 °C for one hour .
At the end of incubation, sacs were drained into gradu-
ated centrifuge tubes (to measure any volume change) and weighed.
Mucosal and
eerosal . solutions were centrifuged to remove sloughed tissue, and asesyed by liquid scintillation counting .
Since the initial condition was the same con-
centration of compound inside and outside of the intestinal eaca, a~ increase in the inside (serosal) concentration in the absence of water lose represented movement against a concentration gradient .
The compounds utilized ware L-
cystine- 3H (Schwarz Bioresearch) and L-djenkolic acid- 3H prepared by a special technique that will ba described in detail in a separate cammunicatioa; the djeakolic acid was essentially entirely in the Inform, aé determined by optical rotation .
Carrier was added as necessary.
Periodic chromatographic checks of
the mucosal and serosal solutions revealed no radioactivity outside of the ra dioactive peak corresponding to Qie authentic compound .
Inhibition studies
were carried out by adding nonlabeled amino acids to the mucoeal and serosal fluids to determine if they interfered with L-djenkolic acid transport . Results In the absence of glucose, water transport by the intestinal eaca was is the mucoeal to aeroeal direction and was negligible
(0-0 .1 ml, gained) .
Hence as increase is radioactivity in the aeroeal fluid represented transport and not increased concentration due to water loan . djeakolic acid of 1 x lÔ
4
At a concentration of Ir
M, when three sate were made from the small intes-
tine of èach hamster, the maximal concentration observed was 1 .04 (aeroeal concastration/standard concentration) with a probable error of 0 .05 due to pipetting and counting .
This is consistent with the previous inability to demonstrate
INTESTINAL TRANSPORT
Vol . S, .No . 13
transport against a concentration gradient .
1165
In an effort to maximize any trans-
port, movement of L-cyetine was next studied as a function of the site of oriResults are shown in Fig. 1 .
gin of the gut sac .
More was transported is the
fifth of six sacs from the hamster small intestine ; approximately 2 .6 times more was transported in the fifth sac than in the first . Accordingly, L-djenkolic acid transport studies were repeated, using only the fifth sac out of six prepared from the hamster's small gut . 1 x lÔ
4
At
M, the ratio of aeroeal concentration/standard concentration was 1.12 .
This was the mean of 12 such sacs ; transport was demonstrable in ten.
40 q 30 ' 0
N
m .-1
20 '.
1
2
3
5
4
Sac Number FIG . 1 Serosal gain of L-cystine (using 1 x lÔ Bach point is the mean of six hamsters .
4
M) .
Recognizing that other species might more readily transport L-djenkolic acid than the hamster, the mouse gut, of 1 x
10
4
the mouse small intestine was also employed .
Using
transport of L-djenkolic acid was demonstrable at coacentrationa
M to 1 x
10
9
M in all sacs .
The maximum ratio of the aeroeal con-
centration/standard concentration was 1.21 at 1 x 10
4
M L-djenkolic acid .
Over the small concentration range open to study (limited at the higher range by insolubility, and at the lower range by the aecesaity for adequate atatiatics of counting),
fully adequate kinetic investigation could not be obtained .
The maximum transport of L-djenkolic acid by the mouse small Lateatiae vas
INTESTINAL TRANSPORT
1166 approximately 0 .3
Vol . 5, No . 13
moles/gm . tissue, while the apparent Michaelie coastant was
on the order of 2 x
10 4
M.
Due to the limited concentration range in which L-djenkolic acid could be utilized, definitive studies on inhibition of transport by other amino acids were not possible .
However,
hibited over 90X by 5 x
10
tion obtainable (under 1 x transport of 1 x lÔ
4
3
transport of 5 x lÔ M L-lysine .
10
3
4
M L-djenkolic acid was in
At the maximum L-cyatine concentra-
M), there was no significant depression of the
M L-djenkolic acid . Discussion
The spatial distribution of L-cyatine movement by the in vitro hamster small intestine is similar (when averaged over several animals) to that of the neutral amino acids, and the amino acida6. reported for the rats .
However,
it differs from that
Utilizing the conventional techniques of three sacs
per hamster, no clear transport of L-djenkolic acid could be demonatrated 4 . However,
by concentrating on the likely site of maximal movement, slight concen-
tration has been shown by the present experiments . analogues of cyatine, previously studied in the D,Lsacs per hamster, lined here .
They suggest that other form and by using three
should be repeated under the special transport conditions out-
That is, the hamster should be fasted, glucose should be absent
from the medium, and the fifth of six intestinal sacs per animal should be employed .
Transport of L-djenkolic acid by the mouse small intestine may serve
to emphasize the observation that this preparation maintains its viability following removal from the body, and gives transport kinetics that approach the in vivo situation$ . Summary The spatial distribution of L-cyatine transport by the in vitro hamster small intestine was described .
More was transported five-sixths of the way
dawn the gut than at other locales .
Using hamster intestinal sacs made from
this area only, in buffer devoid of glucose (to reduce water transport) and L-
Vol .
5, No . 13
djenkolic acid at 1 x
116
INTESTINAL TRANSPORT 10
4
M, miaimal transport against a concentratioa grad-
seat was demoastrable in about 90R of the experiments .
Utiliziag the mouse
small intestine, all sacs transported L-djenkolic acid against a conceatratioa gradient .
Transport was inhibited by L-lysine .
The other D,L- analogues of
cyatine previously noted not to be transported should be re-investigated employing the L-isomers and the conditions outlined here with the hamster gut,
or
preferably with the small intestine of the mouse . RefereACes 1.
L.B . Rosenberg, J.L . Durant and J .M . Holland, New ßiüi . J. Med . 273, (1965) .
1239
2.
S .C .C . Lin, H. Hagihira aad T .H . Wflson, Am . J . Phvaiol. 202, 919 (1962) .
3.
H.G . Randall and D .F . Svered, Biochim. Biouhus . Acta = , 98 (1964) .
4.
R.P . Spencer, K .R . Brody aad H.G . Mautaer, Nature 207, 418 (1965) .
5.
L. Leive aad B .D . Davis, J. Biol . Chem . 240, 4362 (1965) .
6.
R.P . Spencer, K .R . Brody and B.M . Lottere, Biochim. Bioohvs . Acta â8, 400 (1964) .
7.
M.W . Neil, Biochem. J . ~, 118 (1959) .
8.
W. Wright, Fed. Proc . ~, 416 (1966) .