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Studies on the exocrino-enteric circulation of mus musculus
Comp. Biochem. PhysioL, 1970, VoL 32, pp. 55 to 60. PergamonPress. Printed in Great Britain
STUDIES ON THE EXOCRINO-ENTERIC CIRCULATION OF M U S M U S C U L U S R. G. L I T C H F O R D University of Tennessee at Chattanooga,* Chattanooga, Tennessee 37403 (Received 14July 1969)
Abstract--1. The rate of hepatic secretion of intestinally absorbed L-
methionine-methyl-t4C-saline is studied in normally functioning and pathologically altered Mus musculus. 2. Time analyses of ~4Cradiation in the peripheral blood demonstrate, in vivo, the existence of a steady-state system between host and parasite. 3. The effect of parasitism on host physiology is clearly demonstrated by different products of methionine metabolism occurring in the hepatic secretions of parasitized and non-parasitized M. musculus.
INTRODUCTION
RECENT evidence has been offered showing that plasma amino acid ratios, as well as gut amino acid ratios, are regulated at relatively constant levels, especially during periods of high-quality protein intake (Ganapathy & Nasset, 1962; Nasset et al., 1963 ; Nasset, 1965; Olmstead et al., 1966). Amino acids actively transported from the gut lumen pass through the mucosa, which may remove some of them, and into the mesenteric and portal veins and are subjected to selection by the liver before entering the peripheral vessels. Because of this process, as well as general body metabolism, it is not surprising that the levels of the amino acid molar ratios in the gut and in the blood are not equal. Although the involvement of hepatic secretion in the exocrino-enteric circulation is well known, the rates at which this may occur are not well known. Since the ratios of the amino acids are regulated in the gut and the blood stream it should be possible to determine the rate at which a labeled compound would make its appearance in the peripheral blood and in the bile. It would be of interest to determine what effect, if any, pathological conditions associated with the biliary organs of the exocrino-enteric circulatory pathway would have on the rates of movement of compounds into the bile. A natural infection of Hymenolepis microstoma offers just such a pathology. Direct measurements are proposed for the determination of the rate of movement of a compound from the gut to the bile via transport by the blood and secretion by the metabolically active liver of parasitized and non-parasitized mice. * This work was supported by P.H.S. Grants 5TI-AI-106 and AI-01384 under the direction of C. P. Read, Rice University. 55
56
R . G . LITCHFORD
MATERIALS AND METHODS All animals used were 8-9-week-old male mice (Mus rnusculus) of the I C R Swiss Albino strain. Parasitized mice were infected per os with two each of laboratory-reared cysticercoids of Hymenolepis microstoma. Infected mice were used for experimentation when the worms had attained an age of 12 days. All animals were quartered in a controlled environment and fed Purina Lab Chow and water ad lib. A solution of L-methionine-methylJ4C-saline was prepared as follows : a 1"0 ml solution of labeled methionine (sp. ac. of 13"4 m c / m M and measuring 50 /zc/ml in 0"01 N HC1, New England Nuclear) was diluted with balanced saline containing 0"06 N NaHCO3. This gave a solution of p H 7"4 which contained 25/zc of labeled methionine/ml. Parasitized and non-parasitized mice were anesthetized with nembutal injected intraperitoneally. The bile duct was ligatured just anterior to the sphincter of Boyden and cannulated. In each experiment 0'3 ml of the methionine saline was injected into the duodenum just posterior to the duodenal papilla. Collection of bile began immediately after the injection was completed. After each successive 15 rain, up to 45 min, the collected bile was measured to the nearest 0.01 ml, diluted to 2 x the volume with 70% E T O H and stored at - 20°C. At the termination of the collections the samples for each time period were brought to room temperature. Duplicate 10-/zl samples were placed on planchets and diluted with 0"5 ml of 70% E T O H . A standard of 0"01 ml of the injection fluid, diluted to 10 ml, was placed on duplicate planchets in 10-/zl vol. These were also flooded with 0-5 ml of 70% E T O H . All planchets were dried and counted with a gas-flow counter. Following correction for background and dilution, the activity was expressed as counts/min per milliliter of original sample. Parasitized and non-parasitized mice, which were ligatured but not cannulated, were utilized for the collections of peripheral blood. These samples were collected in heparinized vessels from transected tails immediately following intraduodenal injection of 0"3 ml of the methionine saline solution. Different animals were used for each time period which ranged from 5 to 20 min. Each sample volume was diluted 3 x with 70% E T O H and stored at - 20°C. When the blood collections were completed, they were warmed to 4°C and centrifuged. The supernatant from each sample was placed on duplicate planchets in 10-/zl vols. and flooded with 0"5 ml of 70% E T O H . T h e dried planchets were counted with the bile samples. RESULTS AND DISCUSSION F i g u r e 1 p r e s e n t s t h e d a t a f r o m t h e p e r i p h e r a l b l o o d samples. I n t h e n o n p a r a s i t i z e d m i c e 4.2 p e r c e n t of t h e a d m i n i s t e r e d dose was r e c o v e r e d after 20 m i n w h e r e a s in t h e p a r a s i t i z e d m i c e 3.6 p e r cent of t h e a d m i n i s t e r e d d o s e was r e c o v e r e d after 20 m i n . S i n c e n o f u r t h e r t r e a t m e n t was a f f o r d e d t h e b l o o d s a m p l e s , it is n o t k n o w n w h a t c o m p o u n d s are r e p r e s e n t e d b y t h e r e c o r d e d activity. A n a l y s i s o f v a r i a n c e p e r f o r m e d o n t h e s e d a t a s h o w significant differences ( P = 0.05) b e t w e e n t h e t w o g r o u p s at t h e 5 - m i n interval. H o w e v e r , t h e s e differences b e c o m e less significant w i t h t i m e . E x t r a p o l a t i o n s of t h e s e c u r v e s s h o w c o n v e r g e n c e o c c u r r i n g b e t w e e n 30 a n d 45 rain. T h i s s u p p o r t s t h e e x i s t e n c e o f a s t e a d y - s t a t e s y s t e m b e t w e e n h o s t a n d p a r a s i t e in t h e f o l l o w i n g w a y : a h i g h rate o f flow of c o m p o u n d s does exist b e t w e e n p a r a s i t e a n d h o s t ( H o p k i n s & Callow, 1965). H o w e v e r , t h e s e w o r k e r s h a v e d e m o n s t r a t e d loss o f p r e - l o a d e d L-methionine-14C f r o m w o r m s to h o s t ( w h i c h was also s h o w n to b e 90 p e r c e n t u n c h a n g e d b y w o r m m e t a b o l i s m ) . In vitro s t u d i e s b y R e a d a n d c o - w o r k e r s (Read, 1966) h a v e s h o w n t h a t hymenolepis also a c c u m u l a t e s a v a r i e t y o f a m i n o acids. I n a d d i t i o n , t h e i r s t u d i e s h a v e r e v e a l e d v a r i e d effects of
THE EXOCRINO--ENTERIC C I R C U L A T I O N OF M U S M U S C U L U S
57
different parameters on the in vitro efflux of a variety of accumulated amino acids. Studies utilizing physiological molar ratios (Read et aL, 1963) indicate what might be expected in an in vivo situation. The in vivo studies presented here imply that Hymenolepis does accumulate labeled methionine and consequently exchanges this with its environment to the extent that a time-course study shows no difference in radioactivity recovered from the peripheral blood of parasitized or non-parasitized mice.
I.O
%_ x
j~
.E (,D O.f
i ~ /
0-01
• =Non- parasitized • =Parasitized !
I 30
15 Time,
___
rain
FIG. 1. Appearance of 1~C in the peripheral blood of mice following intraduodenal injection of L-methionine-methyl-14C-saline. ALl points represent the average values of two mice. The ranges represent the extreme values. Figures 2 and 3 present the data from the bile of parasitized and non-parasitized mice respectively. An analysis o£ variance shows no significant difference between the parameters. This is perplexing in view of the data received from the peripheral blood. However, the role of methionine as a methyl donor is well known and this could have resulted in a "leveling out" effect on detectable 14C activity in the bile samples. Bile samples from five non-parasitized mice and three parasitized mice which had received 0-3 ml of methionine-14C saline intraduodenally were pooled at 30- and 60-min time intervals. Each pooled sample was deproteinized in 2 vol. of 70%
58
R.G.
LITCHFORD
•,U "~ %
~ •
I
0
4o
o
E
= .-~ .=
~
,leo u!w/sluno 0
• ~ ,.~ q::~
o
~~
E
o
t
2
$ 901 x 1w ~ed u!w/s~,un09
6 o
0
.~
~
THE EXOCRINO--ENTERICCIRCULATIONOF M U S
(9
MUSCULUS
O
O
{3::- ?.-'":: A
B
FIO. 4. Composite two-dimensional chromatoradiograms of labeled compounds in the bile of M . musculus 30 min after intraduodenal injection of L-methioninemethyl-X4C-saline. Section A represents mice infected with H. microstoma. Section B represents non-parasitized mice.
©
% ...-..~
A
B
FIG. 5. Composite two-dimensional chromatoradiograms of labeled compounds in the bile of 21/1. musculus 60 rain after intraduodenal injection of L-methioninemethyl-xtC-saline. Section A represents mice infected with H. microstoma. Section B represents non-parasitized mice.
59
60
R.G. LITCHFORD
E T O H , partitioned against chloroform, dried and taken up in 10% 2-propanol. T h e total volume for each sample was subjected to successive runs of onedimensional ascending paper chromatography using secondary butanol-formic acid-water in the ratio of 75 : 15 : 10. T h e chromatograms were prepared for analysis by a Nuclear-Chicago Actigraph II Strip Counter. T h e results of these analyses demonstrated the presence of different compounds in the bile of parasitized and non-parasitized mice. T o verify this the stripped chromatograms were sectioned and each section was eluted in a high-humidity cabinet. T h e eluate was taken to dryness on Teflon watch glasses and taken up in small aliquots of 10% 2-propanol. T h e samples were subjected to two-dimensional paper chromatography (methanol-pyridine-water, 20 : 1 : 5, and secondary butanol-formic acid-water, 75 : 15 : 0) and the resultant chromatograms were allowed to expose non-screen X-ray film for 9 weeks. Actual compound identification has not been attempted. Figures 4 and 5 are facsimiles of the composite pictures interpreted from the radiograms. It is clearly demonstrated that the metabolic pathway of the methyl group of L-methionine is altered by the presence of the bile duct hymenolepidid H. microstoma. T h e manner by which it is altered is not understood and the design of these experiments does not allow further conclusions. REFERENCES GANAPATHYS. N. • NASSETE. S. (1962) Free amino acids in dog blood and gut contents after feeding meat. J. Nutr. 78, 241-244. HOPKINS C. A. & CALLOWL. L. (1965) Methionine flux between a tapeworm (Hymenolepis diminuta) and its environment. Parasitology 55, 653-666. NASSET E. S. (1965) Role of the digestive system in protein metabolism. Fedn Proc. Fedn Am. Socs exp. Biol. 24, 953-958. NASSETE. S., GANAPATHYS. N. & GOLDSMITHD. P. J. (1963) Amino acids in dog blood and gut contents after feeding zein. J. Nutr. 81,343-347. OLMSTEADW. W., NASSETE. S. & KELLEYM. L. (1966) Amino acids in postprandial gut contents of man..7. Nutr. 90, 291-294. READ C. P. (1966) Nutrition of intestinal helminths. In Biology of Parasites (Edited by SOULSBYE. J. L.). Academic Press, New York. READ C. P., ROTHMnNA. H. & SIMMONSJ. E., JR. (1963) Studies on membrane transport, with special reference to parasite-host integration. In Some Biochemical and Immunological Aspects of Host-Parasite Relationships (Edited by CHENOT. C.), Ann. N. Y. Acad. Sci. 113 (Art. 1), 154-205. Key Word Index--Exocrino-enteric circulation; Hymenolepis microstoma, effects on host hepatic secretion of; steady state of host-parasite relationship; Mus musculus, exocrinoenteric circulation of; L-methionine-methyl-14C, studies in mouse exocrino-enteric circulation.
STUDIES ON THE EXOCRINO-ENTERIC CIRCULATION OF M U S M U S C U L U S R. G. L I T C H F O R D University of Tennessee at Chattanooga,* Chattanooga, Tennessee 37403 (Received 14July 1969)
Abstract--1. The rate of hepatic secretion of intestinally absorbed L-
methionine-methyl-t4C-saline is studied in normally functioning and pathologically altered Mus musculus. 2. Time analyses of ~4Cradiation in the peripheral blood demonstrate, in vivo, the existence of a steady-state system between host and parasite. 3. The effect of parasitism on host physiology is clearly demonstrated by different products of methionine metabolism occurring in the hepatic secretions of parasitized and non-parasitized M. musculus.
INTRODUCTION
RECENT evidence has been offered showing that plasma amino acid ratios, as well as gut amino acid ratios, are regulated at relatively constant levels, especially during periods of high-quality protein intake (Ganapathy & Nasset, 1962; Nasset et al., 1963 ; Nasset, 1965; Olmstead et al., 1966). Amino acids actively transported from the gut lumen pass through the mucosa, which may remove some of them, and into the mesenteric and portal veins and are subjected to selection by the liver before entering the peripheral vessels. Because of this process, as well as general body metabolism, it is not surprising that the levels of the amino acid molar ratios in the gut and in the blood are not equal. Although the involvement of hepatic secretion in the exocrino-enteric circulation is well known, the rates at which this may occur are not well known. Since the ratios of the amino acids are regulated in the gut and the blood stream it should be possible to determine the rate at which a labeled compound would make its appearance in the peripheral blood and in the bile. It would be of interest to determine what effect, if any, pathological conditions associated with the biliary organs of the exocrino-enteric circulatory pathway would have on the rates of movement of compounds into the bile. A natural infection of Hymenolepis microstoma offers just such a pathology. Direct measurements are proposed for the determination of the rate of movement of a compound from the gut to the bile via transport by the blood and secretion by the metabolically active liver of parasitized and non-parasitized mice. * This work was supported by P.H.S. Grants 5TI-AI-106 and AI-01384 under the direction of C. P. Read, Rice University. 55
56
R . G . LITCHFORD
MATERIALS AND METHODS All animals used were 8-9-week-old male mice (Mus rnusculus) of the I C R Swiss Albino strain. Parasitized mice were infected per os with two each of laboratory-reared cysticercoids of Hymenolepis microstoma. Infected mice were used for experimentation when the worms had attained an age of 12 days. All animals were quartered in a controlled environment and fed Purina Lab Chow and water ad lib. A solution of L-methionine-methylJ4C-saline was prepared as follows : a 1"0 ml solution of labeled methionine (sp. ac. of 13"4 m c / m M and measuring 50 /zc/ml in 0"01 N HC1, New England Nuclear) was diluted with balanced saline containing 0"06 N NaHCO3. This gave a solution of p H 7"4 which contained 25/zc of labeled methionine/ml. Parasitized and non-parasitized mice were anesthetized with nembutal injected intraperitoneally. The bile duct was ligatured just anterior to the sphincter of Boyden and cannulated. In each experiment 0'3 ml of the methionine saline was injected into the duodenum just posterior to the duodenal papilla. Collection of bile began immediately after the injection was completed. After each successive 15 rain, up to 45 min, the collected bile was measured to the nearest 0.01 ml, diluted to 2 x the volume with 70% E T O H and stored at - 20°C. At the termination of the collections the samples for each time period were brought to room temperature. Duplicate 10-/zl samples were placed on planchets and diluted with 0"5 ml of 70% E T O H . A standard of 0"01 ml of the injection fluid, diluted to 10 ml, was placed on duplicate planchets in 10-/zl vol. These were also flooded with 0-5 ml of 70% E T O H . All planchets were dried and counted with a gas-flow counter. Following correction for background and dilution, the activity was expressed as counts/min per milliliter of original sample. Parasitized and non-parasitized mice, which were ligatured but not cannulated, were utilized for the collections of peripheral blood. These samples were collected in heparinized vessels from transected tails immediately following intraduodenal injection of 0"3 ml of the methionine saline solution. Different animals were used for each time period which ranged from 5 to 20 min. Each sample volume was diluted 3 x with 70% E T O H and stored at - 20°C. When the blood collections were completed, they were warmed to 4°C and centrifuged. The supernatant from each sample was placed on duplicate planchets in 10-/zl vols. and flooded with 0"5 ml of 70% E T O H . T h e dried planchets were counted with the bile samples. RESULTS AND DISCUSSION F i g u r e 1 p r e s e n t s t h e d a t a f r o m t h e p e r i p h e r a l b l o o d samples. I n t h e n o n p a r a s i t i z e d m i c e 4.2 p e r c e n t of t h e a d m i n i s t e r e d dose was r e c o v e r e d after 20 m i n w h e r e a s in t h e p a r a s i t i z e d m i c e 3.6 p e r cent of t h e a d m i n i s t e r e d d o s e was r e c o v e r e d after 20 m i n . S i n c e n o f u r t h e r t r e a t m e n t was a f f o r d e d t h e b l o o d s a m p l e s , it is n o t k n o w n w h a t c o m p o u n d s are r e p r e s e n t e d b y t h e r e c o r d e d activity. A n a l y s i s o f v a r i a n c e p e r f o r m e d o n t h e s e d a t a s h o w significant differences ( P = 0.05) b e t w e e n t h e t w o g r o u p s at t h e 5 - m i n interval. H o w e v e r , t h e s e differences b e c o m e less significant w i t h t i m e . E x t r a p o l a t i o n s of t h e s e c u r v e s s h o w c o n v e r g e n c e o c c u r r i n g b e t w e e n 30 a n d 45 rain. T h i s s u p p o r t s t h e e x i s t e n c e o f a s t e a d y - s t a t e s y s t e m b e t w e e n h o s t a n d p a r a s i t e in t h e f o l l o w i n g w a y : a h i g h rate o f flow of c o m p o u n d s does exist b e t w e e n p a r a s i t e a n d h o s t ( H o p k i n s & Callow, 1965). H o w e v e r , t h e s e w o r k e r s h a v e d e m o n s t r a t e d loss o f p r e - l o a d e d L-methionine-14C f r o m w o r m s to h o s t ( w h i c h was also s h o w n to b e 90 p e r c e n t u n c h a n g e d b y w o r m m e t a b o l i s m ) . In vitro s t u d i e s b y R e a d a n d c o - w o r k e r s (Read, 1966) h a v e s h o w n t h a t hymenolepis also a c c u m u l a t e s a v a r i e t y o f a m i n o acids. I n a d d i t i o n , t h e i r s t u d i e s h a v e r e v e a l e d v a r i e d effects of
THE EXOCRINO--ENTERIC C I R C U L A T I O N OF M U S M U S C U L U S
57
different parameters on the in vitro efflux of a variety of accumulated amino acids. Studies utilizing physiological molar ratios (Read et aL, 1963) indicate what might be expected in an in vivo situation. The in vivo studies presented here imply that Hymenolepis does accumulate labeled methionine and consequently exchanges this with its environment to the extent that a time-course study shows no difference in radioactivity recovered from the peripheral blood of parasitized or non-parasitized mice.
I.O
%_ x
j~
.E (,D O.f
i ~ /
0-01
• =Non- parasitized • =Parasitized !
I 30
15 Time,
___
rain
FIG. 1. Appearance of 1~C in the peripheral blood of mice following intraduodenal injection of L-methionine-methyl-14C-saline. ALl points represent the average values of two mice. The ranges represent the extreme values. Figures 2 and 3 present the data from the bile of parasitized and non-parasitized mice respectively. An analysis o£ variance shows no significant difference between the parameters. This is perplexing in view of the data received from the peripheral blood. However, the role of methionine as a methyl donor is well known and this could have resulted in a "leveling out" effect on detectable 14C activity in the bile samples. Bile samples from five non-parasitized mice and three parasitized mice which had received 0-3 ml of methionine-14C saline intraduodenally were pooled at 30- and 60-min time intervals. Each pooled sample was deproteinized in 2 vol. of 70%
58
R.G.
LITCHFORD
•,U "~ %
~ •
I
0
4o
o
E
= .-~ .=
~
,leo u!w/sluno 0
• ~ ,.~ q::~
o
~~
E
o
t
2
$ 901 x 1w ~ed u!w/s~,un09
6 o
0
.~
~
THE EXOCRINO--ENTERICCIRCULATIONOF M U S
(9
MUSCULUS
O
O
{3::- ?.-'":: A
B
FIO. 4. Composite two-dimensional chromatoradiograms of labeled compounds in the bile of M . musculus 30 min after intraduodenal injection of L-methioninemethyl-X4C-saline. Section A represents mice infected with H. microstoma. Section B represents non-parasitized mice.
©
% ...-..~
A
B
FIG. 5. Composite two-dimensional chromatoradiograms of labeled compounds in the bile of 21/1. musculus 60 rain after intraduodenal injection of L-methioninemethyl-xtC-saline. Section A represents mice infected with H. microstoma. Section B represents non-parasitized mice.
59
60
R.G. LITCHFORD
E T O H , partitioned against chloroform, dried and taken up in 10% 2-propanol. T h e total volume for each sample was subjected to successive runs of onedimensional ascending paper chromatography using secondary butanol-formic acid-water in the ratio of 75 : 15 : 10. T h e chromatograms were prepared for analysis by a Nuclear-Chicago Actigraph II Strip Counter. T h e results of these analyses demonstrated the presence of different compounds in the bile of parasitized and non-parasitized mice. T o verify this the stripped chromatograms were sectioned and each section was eluted in a high-humidity cabinet. T h e eluate was taken to dryness on Teflon watch glasses and taken up in small aliquots of 10% 2-propanol. T h e samples were subjected to two-dimensional paper chromatography (methanol-pyridine-water, 20 : 1 : 5, and secondary butanol-formic acid-water, 75 : 15 : 0) and the resultant chromatograms were allowed to expose non-screen X-ray film for 9 weeks. Actual compound identification has not been attempted. Figures 4 and 5 are facsimiles of the composite pictures interpreted from the radiograms. It is clearly demonstrated that the metabolic pathway of the methyl group of L-methionine is altered by the presence of the bile duct hymenolepidid H. microstoma. T h e manner by which it is altered is not understood and the design of these experiments does not allow further conclusions. REFERENCES GANAPATHYS. N. • NASSETE. S. (1962) Free amino acids in dog blood and gut contents after feeding meat. J. Nutr. 78, 241-244. HOPKINS C. A. & CALLOWL. L. (1965) Methionine flux between a tapeworm (Hymenolepis diminuta) and its environment. Parasitology 55, 653-666. NASSET E. S. (1965) Role of the digestive system in protein metabolism. Fedn Proc. Fedn Am. Socs exp. Biol. 24, 953-958. NASSETE. S., GANAPATHYS. N. & GOLDSMITHD. P. J. (1963) Amino acids in dog blood and gut contents after feeding zein. J. Nutr. 81,343-347. OLMSTEADW. W., NASSETE. S. & KELLEYM. L. (1966) Amino acids in postprandial gut contents of man..7. Nutr. 90, 291-294. READ C. P. (1966) Nutrition of intestinal helminths. In Biology of Parasites (Edited by SOULSBYE. J. L.). Academic Press, New York. READ C. P., ROTHMnNA. H. & SIMMONSJ. E., JR. (1963) Studies on membrane transport, with special reference to parasite-host integration. In Some Biochemical and Immunological Aspects of Host-Parasite Relationships (Edited by CHENOT. C.), Ann. N. Y. Acad. Sci. 113 (Art. 1), 154-205. Key Word Index--Exocrino-enteric circulation; Hymenolepis microstoma, effects on host hepatic secretion of; steady state of host-parasite relationship; Mus musculus, exocrinoenteric circulation of; L-methionine-methyl-14C, studies in mouse exocrino-enteric circulation.