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Intestinal absorption and metabolism of prostaglandin E1−1−14C by thoracic duct and bile duct cannulated rats
Life Sciences Vol. 7, Part I, pp. 883-889, 1988. Printed in Great Britain .
Pergamon Press
INPESTINAL ABSORPTION AND METABOLISM OF PROSTAGLANDIN Ey-1-14C BY THORACIC DUCT AND BILE DUCP CANNULATED RATS T . M. Parkinson, .J . C . Schneider, Jr ., J . J . Krake and W. L. Miller Metabolic Diseases Research, The Upjohn Compar~y, Kalamazoo, Michigan
b9001
(Received 3 Niay 1968; in final form 7 June 1968) ALTHOUGH several studies have been published on the metabolism of parenterally administered 14C- or 3Ii-labelled prostaglandin E1
(PGEy), corresponding data
on the metabolism of orally administered compound have not been reported previously .
20
Samuelsson (1) foiu.d that
PGE1-5,6-3Ii
hours after intravenous infusion of
to rats, about 50~ of the isotope was recovered in the urine as
more polar metabolites and 10~, was recovered in the feces .
In man, after
intravenous administration, 3Ii-labelled PGEl disappeared rapidly from the blood and was excreted in the urine
(60~)
and feces
(40y6) (2) .
When PGEy-1-C1~ was
constantly infused into fed rats via an indwelling jugular tannins, ity was exhaled as 14C0 2 at a maximum rate of
4996
radioactiv-
of the infusion rate ; in
animals fasted for 15 hours prior to dosing, rate of expired 14 C0 2 production was
68',b
of the infusion rate (3) .
About
recovered in the urine of a fed rat in
72
7-8'~6
of the infused radioactivity was
hours (3) .
In the present study rata
with thoracic duct or bile duct cannulas housed in a metabolic chamber were given FGE1-1-14 C dissolved in cottonseed oil orally via stomach tube and 14C02 in expired air as well as radioactivity in lymph or bile were measured over the next
24
hours . Materials and Methods
Thoracic ducts of fed male rats
(Sprague-Dawley, Upjohn strain) weighing
approximately 300 g were cannulated with polyethylene tubing (Intramedic PE
60,
Clay-Adams, Inc ., New York) using modifications of the procedure of Bollman et al . (4) .
Animals were placed in restraining cages without food but with free
883
Vol. 7, No . 15
PROSTAGLANDIN
884
access to 0 .996 saline and 16-20 hours later were given by s1_omach tube 0 .7-1 .0 ml cottonseed oil (Wesson Sales Co ., Fullerton, Calif .) containing 1 .2~ WC PGE1-1- 14 C per ml (specific activity
WC~mg)(5) .
j .~Fl
The animals were placed
in a glass metabolic chamber (Aerospace Industries, Inc ., Garnersville, N . Y .) and the chamber was connected to a Bio-Monitor (Beckma.n Instruments, Inc ., Medical Systems Operations, Fullerton, Calif .) which measured total exhaled C02, total 14C02, and calculated the specific activity of exhaled C02 .
Total
C02 was measured by a thermal conductivity detector and radioactive C0~ was measured by a vibrating-reed electrometer .
Lsymph was collected at l, 3, j, 12
and 24 hours in glass tubes containing 0 .5 ml of heparin in saline (Upjohn, 1000 units~ml) .
One-tenth ml of lymph from each collection period was counted
in 10 ml of naphthalene-dioxane scintillation fluid (6) in a Tri Carb (Packard Instrument Sales Corp ., Cleveland, Ohio) scintillation spectrometer .
Absolute
sample activities were determined using a toluene- 14 C internal standard .
Ali-
quots of 24-hour pooled Lymph collections were extracted with 1-butanol or 80$ aqueous ethanol (1) and extracts were chromatographed on Silica Gel G plates in chloroform-methanol-acetic acid (90 :5 :5) .
Spots were detected by heating
the plates to 120 ° and spraying with 1096 phosphomolybdic acid in absolute ethanol and were scraped off the plates into vials and counted in 10 ml diotol scintillation fluid (7) . Results Average specific activity in expired air for two rats in shown in Fig . 1 . Radioactivity appeared in the breath rapidly after dosing and maximum specific activity was measured within 50-40 minutes .
Specific activity then fell so
rapidly that by the end of hour 2 the initial peak was complete and accounted for about 15$ of the administered dose .
A second peak began to form between
hour 3 and hour 4 and reached a maximum by hour 7.
This second peak was com-
plete by hours 10-12 and contained about 1~+~, of the administered radioactivity . Peaks 1 and 2 accounted For about >096 of the total expired l 'C02, or about 2~
Vol. 7, No . 15
PROSTAGLANDIN
885
FIG . 1
40
35
N U N U a
30
25
z r
20
a
15
U
U W a N
10
5
Q lvvv
0
2
.
4
.vvv
6
8
10
W-
12
16 20 HOURS AFTER ORAL DOSE OF PGE~-I-~ 4 C
%rcretion of 14 C0~ irr "_xpix~e:l air aftc?~ oral administration of PGLl-1- l ''C '. .o thoracic tract car:nuJcrtc " d rats . Data are the at~era.gen cf two r~ri .mals . cl' t. } :e administered dosa .
!üt additio?ra.l : ;,x!~ of '. : :re dose "aas accounted for by a
rat.lter con;i.ara. rate of ~"xcretion of l''CC ; : trp to :rct:rc l'(-20 .
Radioactivity in
2'a-hour ly-tnplr scruples accounted for G .'i-i .0~~ ci' ±!,~, adn:ir:ist .ei~ed donc" (Table 1) .
Specific activil.y of collF_cte.3 iy?nç~a (dhr::/n.l .
react:c d a rna:timu:n ~-12 trouts
after dosing anà remained relaatinely con tan' . dr.rir :ë, n.l :e rest of tl .v collection period .
'TLC of ly???plr ext!~ac+. :~ d~_tec ; .e~i r:o ccr??por :+ :rrts wi`.I : ' . .,_ Mine Rf. a~ PGE1
or PG/11 star~dar :ds ci .rcn?a.tol :ral~:,~ :~: ::ir. :ua~:arx ;r ; : . .!y .
i~:l .i" r~ ao PGE1 xnd PCA1 t,av~
886
PROSTAGLANDIN
U
ro
rl
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tD
i+ q N rl
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~
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.?
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Vol . 7, No. 15
N r-I -?
L
N I
L
~n N y 3
F+ rn
O
~
O
:7~
rl
N
N
O~ -7
I a
ro N r-I m
m ti 0
v~
N
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rl O
~+ rl
ô
~O
O
a Si
,i ,~ U O "rl
ro
O r-I
N
Kl
N rl
ai +~
H
PR06TAGLANDIN
Vol. 7, No . 15
887
Rf values in this system of 0 .35 and 0 .70, respectively, 95% of the radioactivity recovered from the TLC plates remained at origin, suggesting that 14 C from PGEl-1- 14C may have been incorporated into non-prostaglandin metabolites . Samuelsson (1) reported that rats given PGE1- 3fi intravenously excreted about 10~, of the radioactivity in the bile within 5 hours after dosing .
It
seemed possible, therefore, that peak 2 in expired 14 C0z could result from absorption and metabolism of radioactive products in the enterohepatic ciroulation derived from PGE1-1- 14C .
Expired C02 was analyzed from a rat given PGE1-
1-14C orally after bile duct cannulation .
Peak 2 did not occur in the 14 C02
profile of this animal ; 6~ of the administered dose of radioactivity was recovered in the bile in 24 hours (Fig . 2) .
A sample of the pooled 24-hour bile
FIG . 2
8
z ~~ o ,= 4
F E W Ô
X ~ W
~ 2 d
d
1/ L 1
0
2
1
1
4
1
1
6
1
1
8
1
1
1
1
1
10
12
16
20
24
HOURS AFTER ORAL DOSE OF PGE~-I-~ 4C
Excretion of 14 carbon in bile after oral administration of 1 .07 WC PGE1-1- 14 C . Bile samples (0 .05 ml) were counted in 10 ml naphthalene-dioxane scintillation fluid . collection was extracted with 1-butanol and the residue was chromatographed on Silica Gel G as described for lymph extracts . or PGA1 were detected .
No spots corresponding to PGE 1
Eighty-eight per cent of the radioactivity recovered
From the TLC plate remained at origin, flzrther suggesting incorporation into
PROSTAGLANDIN
888
Vol . 7, No . 15
non-prostaglandin metabolites . Discussion Miller and Krake (3) reported previously that PGEl-1- 14C was rapidly and extensively metabolized to 14 C02 during intravenous infusion .
In the present
study similar rapid excretion of radioactivity in expired air is seen after oral administration .
Harnberg and Samuelsson (8) ha "re found that rat liver
mitochondria degrade PGEl~and PGF ycx in vitro to their Cls homologs by one ßoxidation sequence .
Isolation of the Cla homolog of PGFy~ from urine after
administering the parent compound subcutaneously to rats (y) indicates that the prostaglandins are oxidized via this same pathway in vivo, as previously suggested by Granström, Inger and Samuelsson (9) .
Thus it 1s possible that PGFl-
1- 14 C given orally to thoracic duct cannulated rats was absorbed, carried to the liver in the portal blood, and subjected to ß-oxidation .
Acetate-l- l ~C
formed in these reactions could be oxidized further via the citric acid cycle to 14C02 or be incorporated into other metabolites .
However, the rapidity with
which radioactive C0 2 appeared in the breath after oral administration, and the presence of 14 C-labelled lipids in lymph also suggests the possibility that ßoxidation of PGE1 may take place in the intestine itself .
Indeed, preliminary
studies indicate that 14 C02 is produced by rat jejunum perfused with PGEl-1- 14 C in vitro and that radioactivity is incorporated into both the saponifiable and non-saponifiable fractions of tissue lipids (Parkinson and Schneider, to be published) . Summary Approximately 58',~ of a dose of PGE1-1- 14 C administered orally to thoracic duct cannulated rats was excreted as 1 ~C02 in expired air in 24 hours ; an additional O .j-5 .0$ was found in thoracic duct lymph .
About ~0~6 of the radio
activity in expired air was accounted for in two peaks of maximum specific activity occurring 30-ü0 minutes and ~-7 hours after dosüig .
The second peak
PROSTAGLAI~IN
Vol. 7, No. 15
889
did not occur in the 14 C02 profile of a bile duct cannulated rat and may represent metabolism of radioactive products in the enterohepatic circulation derived from PGE1-1-14 C . References
~, 4091 (1964) .
1.
B . SAMUEISSON, J . Biol . Chem .
2.
E . GRANSTROM, Progr . Biochem. Pharmacol.
3.
W . L . MILLER and J . J . KRAKE, Federation Proc .
4.
J . L . BOLLMAN, J . C . CAIN and J . A. GRINDLAY, J . Isb . Clin . Med .
5.
W . P. SCHNEIIIER, J . E . PIKE and F . P . KUPIECKI, Biothun . Biophys . Acta
6.
J . A . BRAY, Anal . Biochez
1,
7.
R . J . HARBERG, Anal . Chem .
~2, 42 (1960) .
8. 9.
~, 89 (1967) . ~, 241 (1968) .
(1948) .
~, 1349
611 (1966) .
279 (1960) .
M. HAMBERG and B . SAMUEISSON, to be presented before the Federation of European Biochemical Societies, Prague, July, 1968 . a E . GRANSTROM, V. INGER and B . SAMUELSSON, J . Biol . Chem . 240,
457 (1965) .
Pergamon Press
INPESTINAL ABSORPTION AND METABOLISM OF PROSTAGLANDIN Ey-1-14C BY THORACIC DUCT AND BILE DUCP CANNULATED RATS T . M. Parkinson, .J . C . Schneider, Jr ., J . J . Krake and W. L. Miller Metabolic Diseases Research, The Upjohn Compar~y, Kalamazoo, Michigan
b9001
(Received 3 Niay 1968; in final form 7 June 1968) ALTHOUGH several studies have been published on the metabolism of parenterally administered 14C- or 3Ii-labelled prostaglandin E1
(PGEy), corresponding data
on the metabolism of orally administered compound have not been reported previously .
20
Samuelsson (1) foiu.d that
PGE1-5,6-3Ii
hours after intravenous infusion of
to rats, about 50~ of the isotope was recovered in the urine as
more polar metabolites and 10~, was recovered in the feces .
In man, after
intravenous administration, 3Ii-labelled PGEl disappeared rapidly from the blood and was excreted in the urine
(60~)
and feces
(40y6) (2) .
When PGEy-1-C1~ was
constantly infused into fed rats via an indwelling jugular tannins, ity was exhaled as 14C0 2 at a maximum rate of
4996
radioactiv-
of the infusion rate ; in
animals fasted for 15 hours prior to dosing, rate of expired 14 C0 2 production was
68',b
of the infusion rate (3) .
About
recovered in the urine of a fed rat in
72
7-8'~6
of the infused radioactivity was
hours (3) .
In the present study rata
with thoracic duct or bile duct cannulas housed in a metabolic chamber were given FGE1-1-14 C dissolved in cottonseed oil orally via stomach tube and 14C02 in expired air as well as radioactivity in lymph or bile were measured over the next
24
hours . Materials and Methods
Thoracic ducts of fed male rats
(Sprague-Dawley, Upjohn strain) weighing
approximately 300 g were cannulated with polyethylene tubing (Intramedic PE
60,
Clay-Adams, Inc ., New York) using modifications of the procedure of Bollman et al . (4) .
Animals were placed in restraining cages without food but with free
883
Vol. 7, No . 15
PROSTAGLANDIN
884
access to 0 .996 saline and 16-20 hours later were given by s1_omach tube 0 .7-1 .0 ml cottonseed oil (Wesson Sales Co ., Fullerton, Calif .) containing 1 .2~ WC PGE1-1- 14 C per ml (specific activity
WC~mg)(5) .
j .~Fl
The animals were placed
in a glass metabolic chamber (Aerospace Industries, Inc ., Garnersville, N . Y .) and the chamber was connected to a Bio-Monitor (Beckma.n Instruments, Inc ., Medical Systems Operations, Fullerton, Calif .) which measured total exhaled C02, total 14C02, and calculated the specific activity of exhaled C02 .
Total
C02 was measured by a thermal conductivity detector and radioactive C0~ was measured by a vibrating-reed electrometer .
Lsymph was collected at l, 3, j, 12
and 24 hours in glass tubes containing 0 .5 ml of heparin in saline (Upjohn, 1000 units~ml) .
One-tenth ml of lymph from each collection period was counted
in 10 ml of naphthalene-dioxane scintillation fluid (6) in a Tri Carb (Packard Instrument Sales Corp ., Cleveland, Ohio) scintillation spectrometer .
Absolute
sample activities were determined using a toluene- 14 C internal standard .
Ali-
quots of 24-hour pooled Lymph collections were extracted with 1-butanol or 80$ aqueous ethanol (1) and extracts were chromatographed on Silica Gel G plates in chloroform-methanol-acetic acid (90 :5 :5) .
Spots were detected by heating
the plates to 120 ° and spraying with 1096 phosphomolybdic acid in absolute ethanol and were scraped off the plates into vials and counted in 10 ml diotol scintillation fluid (7) . Results Average specific activity in expired air for two rats in shown in Fig . 1 . Radioactivity appeared in the breath rapidly after dosing and maximum specific activity was measured within 50-40 minutes .
Specific activity then fell so
rapidly that by the end of hour 2 the initial peak was complete and accounted for about 15$ of the administered dose .
A second peak began to form between
hour 3 and hour 4 and reached a maximum by hour 7.
This second peak was com-
plete by hours 10-12 and contained about 1~+~, of the administered radioactivity . Peaks 1 and 2 accounted For about >096 of the total expired l 'C02, or about 2~
Vol. 7, No . 15
PROSTAGLANDIN
885
FIG . 1
40
35
N U N U a
30
25
z r
20
a
15
U
U W a N
10
5
Q lvvv
0
2
.
4
.vvv
6
8
10
W-
12
16 20 HOURS AFTER ORAL DOSE OF PGE~-I-~ 4 C
%rcretion of 14 C0~ irr "_xpix~e:l air aftc?~ oral administration of PGLl-1- l ''C '. .o thoracic tract car:nuJcrtc " d rats . Data are the at~era.gen cf two r~ri .mals . cl' t. } :e administered dosa .
!üt additio?ra.l : ;,x!~ of '. : :re dose "aas accounted for by a
rat.lter con;i.ara. rate of ~"xcretion of l''CC ; : trp to :rct:rc l'(-20 .
Radioactivity in
2'a-hour ly-tnplr scruples accounted for G .'i-i .0~~ ci' ±!,~, adn:ir:ist .ei~ed donc" (Table 1) .
Specific activil.y of collF_cte.3 iy?nç~a (dhr::/n.l .
react:c d a rna:timu:n ~-12 trouts
after dosing anà remained relaatinely con tan' . dr.rir :ë, n.l :e rest of tl .v collection period .
'TLC of ly???plr ext!~ac+. :~ d~_tec ; .e~i r:o ccr??por :+ :rrts wi`.I : ' . .,_ Mine Rf. a~ PGE1
or PG/11 star~dar :ds ci .rcn?a.tol :ral~:,~ :~: ::ir. :ua~:arx ;r ; : . .!y .
i~:l .i" r~ ao PGE1 xnd PCA1 t,av~
886
PROSTAGLANDIN
U
ro
rl
q O N +~ N
U o0
tD
i+ q N rl
N1
~
O .-i
lf\
ti
ti
0 q 0 *-I +~
ô
m ,~ q ,-I Fi
O
.?
N
N rl
fi N
O~
L In
~
~
O
U +> U ,-1 U mfi O W O
â q ,i ro
N 0 U
~i
U
fi N r-I I In
a
rn Ô
~+l ro
tn m Fa
m ro
v U
N
Q
m
O
a~
w
ro N
Vol . 7, No. 15
N r-I -?
L
N I
L
~n N y 3
F+ rn
O
~
O
:7~
rl
N
N
O~ -7
I a
ro N r-I m
m ti 0
v~
N
W
rl O
~+ rl
ô
~O
O
a Si
,i ,~ U O "rl
ro
O r-I
N
Kl
N rl
ai +~
H
PR06TAGLANDIN
Vol. 7, No . 15
887
Rf values in this system of 0 .35 and 0 .70, respectively, 95% of the radioactivity recovered from the TLC plates remained at origin, suggesting that 14 C from PGEl-1- 14C may have been incorporated into non-prostaglandin metabolites . Samuelsson (1) reported that rats given PGE1- 3fi intravenously excreted about 10~, of the radioactivity in the bile within 5 hours after dosing .
It
seemed possible, therefore, that peak 2 in expired 14 C0z could result from absorption and metabolism of radioactive products in the enterohepatic ciroulation derived from PGE1-1- 14C .
Expired C02 was analyzed from a rat given PGE1-
1-14C orally after bile duct cannulation .
Peak 2 did not occur in the 14 C02
profile of this animal ; 6~ of the administered dose of radioactivity was recovered in the bile in 24 hours (Fig . 2) .
A sample of the pooled 24-hour bile
FIG . 2
8
z ~~ o ,= 4
F E W Ô
X ~ W
~ 2 d
d
1/ L 1
0
2
1
1
4
1
1
6
1
1
8
1
1
1
1
1
10
12
16
20
24
HOURS AFTER ORAL DOSE OF PGE~-I-~ 4C
Excretion of 14 carbon in bile after oral administration of 1 .07 WC PGE1-1- 14 C . Bile samples (0 .05 ml) were counted in 10 ml naphthalene-dioxane scintillation fluid . collection was extracted with 1-butanol and the residue was chromatographed on Silica Gel G as described for lymph extracts . or PGA1 were detected .
No spots corresponding to PGE 1
Eighty-eight per cent of the radioactivity recovered
From the TLC plate remained at origin, flzrther suggesting incorporation into
PROSTAGLANDIN
888
Vol . 7, No . 15
non-prostaglandin metabolites . Discussion Miller and Krake (3) reported previously that PGEl-1- 14C was rapidly and extensively metabolized to 14 C02 during intravenous infusion .
In the present
study similar rapid excretion of radioactivity in expired air is seen after oral administration .
Harnberg and Samuelsson (8) ha "re found that rat liver
mitochondria degrade PGEl~and PGF ycx in vitro to their Cls homologs by one ßoxidation sequence .
Isolation of the Cla homolog of PGFy~ from urine after
administering the parent compound subcutaneously to rats (y) indicates that the prostaglandins are oxidized via this same pathway in vivo, as previously suggested by Granström, Inger and Samuelsson (9) .
Thus it 1s possible that PGFl-
1- 14 C given orally to thoracic duct cannulated rats was absorbed, carried to the liver in the portal blood, and subjected to ß-oxidation .
Acetate-l- l ~C
formed in these reactions could be oxidized further via the citric acid cycle to 14C02 or be incorporated into other metabolites .
However, the rapidity with
which radioactive C0 2 appeared in the breath after oral administration, and the presence of 14 C-labelled lipids in lymph also suggests the possibility that ßoxidation of PGE1 may take place in the intestine itself .
Indeed, preliminary
studies indicate that 14 C02 is produced by rat jejunum perfused with PGEl-1- 14 C in vitro and that radioactivity is incorporated into both the saponifiable and non-saponifiable fractions of tissue lipids (Parkinson and Schneider, to be published) . Summary Approximately 58',~ of a dose of PGE1-1- 14 C administered orally to thoracic duct cannulated rats was excreted as 1 ~C02 in expired air in 24 hours ; an additional O .j-5 .0$ was found in thoracic duct lymph .
About ~0~6 of the radio
activity in expired air was accounted for in two peaks of maximum specific activity occurring 30-ü0 minutes and ~-7 hours after dosüig .
The second peak
PROSTAGLAI~IN
Vol. 7, No. 15
889
did not occur in the 14 C02 profile of a bile duct cannulated rat and may represent metabolism of radioactive products in the enterohepatic circulation derived from PGE1-1-14 C . References
~, 4091 (1964) .
1.
B . SAMUEISSON, J . Biol . Chem .
2.
E . GRANSTROM, Progr . Biochem. Pharmacol.
3.
W . L . MILLER and J . J . KRAKE, Federation Proc .
4.
J . L . BOLLMAN, J . C . CAIN and J . A. GRINDLAY, J . Isb . Clin . Med .
5.
W . P. SCHNEIIIER, J . E . PIKE and F . P . KUPIECKI, Biothun . Biophys . Acta
6.
J . A . BRAY, Anal . Biochez
1,
7.
R . J . HARBERG, Anal . Chem .
~2, 42 (1960) .
8. 9.
~, 89 (1967) . ~, 241 (1968) .
(1948) .
~, 1349
611 (1966) .
279 (1960) .
M. HAMBERG and B . SAMUEISSON, to be presented before the Federation of European Biochemical Societies, Prague, July, 1968 . a E . GRANSTROM, V. INGER and B . SAMUELSSON, J . Biol . Chem . 240,
457 (1965) .