Bile Acid Pool in Wistar Rats M.M. FISHER, Departments of1 Medicine and Pathology, G. KAKIS, and I.M. YOUSEF, Department of Pathology, University of Toronto, Toronto, Ontario, Canada M5S 1A8
studied. Twenty-four animals, 12 male and 12 female, were kept in a constant temperature The bile acid pool was found to be ca. environment (22 C) in darkness from 7 p.m. to 50 /amoles/100 g body wt in male and 7 a.m. and allowed water ad libitum. One week female rats maintained o n standard laborafter admission to the animal facilities, six male atory chow and ca. 30 #moles/100 g and six female animals were started on a b o d y wt in those maintained on a powdered semisynthetic diet (Teklad Mills, standard semisynthetic diet. The distribuTD-72460 Basal Diet: casein 27%, sucrose tion of bile acids within the pool was 63.9%, corn oil 5%, salt mixture USP XIV 4%, similar in plasma, liver, and intestinal and vitamin mixture 0.1%). Three weeks after tract, except for a higher concentration admission to the animal facility and after an of deoxycholic acid in the intestinal tract. 18-hr fast, the animals were sacrificed by Sex differences in bile acid composition exsanguination under ether anesthesia. were found to be influenced by the dietAs much blood as possible was removed via ary regimen of the animals. the aorta, the intestinal tract was removed after ligation of the distal esophagus and rectum, and INTRODUCTION the liver was removed after ligation of the comThe bile acid pool of the rat has been esti- mon bile duct. Plasma was obtained by centrimated by analysis of the bile acids of the intes- fugation of the blood at 3,000 x g for 20 rain at tinal contents (1), of the bile produced after 4 C. The intestinal tract and the liver were each creation of a biliary fistula (2,3), and of the homogenized vigorously in normal saline, 2 portal blood (4). It has also been estimated ml/g tissue, using a close fitting Teflon-glass more indirectly by isotopic means (5,6). The homo genizer. To 5 ml of plasma, and to aliquots of the pool size in these studies has ranged from ca. 12 to 25 gmoles bile acid/100 g body wt (7,8). intestinal tract and liver homogenates, were From studies on the feedback regulation of bile added tracer amounts of cholyl[ 1-14C] glycine acid synthesis in the rat, Shefer et al. (9) have (CA[1-14C]G) (Amersham Searle, Arlington estimated that the pool of cholyl-taurine in Heights, IL, sp act 49.4 mCi/mmole) and a male rats is ca. 35-40 #moles/100 g body wt. known amount of 5/3-cholanic acid (Applied These workers suggested that previous estimates Science Laboratories, Inc., State College, PA). of the size of the bile acid pool were too low Portions of the plasma and tissue homogenates and that this might explain previous failures to were then extracted with 20 volumes of 0.1% demonstrate a regulatory effect of bile acids on a m m o n i u m hydroxide in absolute ethanol for 4 hr at 60 C. The supernatant obtained after cenbile acid biosynthesis (10). Because of the controversial data in the liter- trifugation at 3,000 x Z for 30 rain at 4 C was saved and this extraction procedure repeated ature and because such information was essential for the interpretation of other investi- twice. The three ethanol extracts were comgations to be conducted in our laboratory, we bined and evaporated to dryness under nitrogen undertook a direct analysis of the size of the after aliquots were taken for radioactivity analbile acid pool in the rat. Because this pool can ysis. Studies of the radioactivity of these exvary with the sex (11) and nutritional status tracts showed an average recovery of 90% of (12) of the animal and because our experi- the added CA[ 1-14C] G. The residue was taken mental program involved the use of a well- up in 5N NaOH and subjected to hydrolysis at defined but semisynthetic diet, we studied both 115 C overnight in sealed tubes. The neutral m a l e a n d female rats on both standard lipids were removed by three extractions of the alkali solution with petroleum ether at 30-60 C laboratory chow and the semisynthetic diet. and, after acidification on ice with 2.5N HC1 to pH 1, the acidic steroids were removed by reMETHODS peated extractions with diethyl ether. The Wistar rats obtained from High Oak Ranch diethyl ether extracts were pooled, checked for (Toronto, Canada) and weighing ca. 200 g were r a d i o a c t i v i t y , and then evaporated under nitrogen. Previous studies in our laboratory 1Yousef is a Canadian Hepatic Foundation Scholar. confirmed that these conditions of alkaline ABSTRACT
93
M.M. FISHER, G. KAKIS, AND I.M. YOUSEF
94
TABLE I Distribution of Bile Acid Pool in Wistar Rats a Purina Chow diet Male Female Intestinal tract (umole/lO0 g body wt) Liverb (gmole/g liver) Blood (lumole/100 ml plasma)
Semisynthetic diet Male Female
50.40 +- 5.10
51.6 :~ 5.40
29.30 + 0.60
27.8 -+ 1.0
0.19 ~ 0.01
0.33 :t 0.01
0.18 -+0.01
0.19 + 0.01
2.96 -+ 0.24
2.97 +- 0.22
1.99•
1.97+-0.12
aValues: mean -+standard error; six animals/group. bLiver wt (g): male, 9.3 +- 0.5; Female, 8.1 +- 0.4 (no significant difference). hydrolysis were equally effective for both glycine and taurine conjugated bile acids (1 I). The completeness of the laydrolysis was indicated by the absence of 14C_radioactivity in the diethyl ether extracts and of CA[1-14C]G in the aqueous layer. The bile acids were methylated, and the trifluoroacetate and trimethyl silyl derivatives of the methylated bile acids were analyzed by gas chromatography as previously described (11,13). The final concentrations of bile acid were calculated on the basis of reference to the internal standard of 5/3cholanic acid (11). Student's t test was used for the statistical analysis, with P > 0.05 indicating no significant difference. RESULTS
The bile acid content of the intestinal tract, blood, and liver of the rats in these studies is presented in Table I. It can be calculated from the data that the total bile acid pool was ca. 100 /~moles in those animals on the Purina Chow diet and 60 ~moles in those on the semisynthetic diet. The reduction in pool size associated with the semisynthetic diet was reflected to an equal degree in the blood, liver, and intestinal tracts. It is apparent that 97% of the bile acid pool is located in the intestinal tract, regardless of sex or diet. There was no sex difference in the size of the bile acid pool in these animals, nor in the concentration of bile acids in the intestinal tract or blood. However, the female rats did have a significantly higher concentration of bile acids in the liver (P < 0.02). The bile acid composition of the intestinal tract, blood, and liver is presented in Table II. Cholic acid (CA) comprised 55-73% o f the bile acid pool. In the intestinal tract, the relative concentrations of CA, lithocholic acid (LCA), and, in those receiving the semisynthetic diet, of chenodeoxycholic acid (CDCA) were signifiLIPIDS, VOL. 11, NO. 2
cantly higher (P < 0.02) in the female rats. The relative concentrations of deoxycho/ic acid ( D O C A ) , /3-muricholic acid (/3MCA), and hyodeoxycholic acid (HyoDOCA) were significantly higher (P < 0.05) in the male. Diet did not appear to influence the relative bile acid composition of the intestinal tract of male rats. Although female rats on the laboratory chow diet appeared to have relatively more CA and relatively less CDCA than did female rats on the semisynthetic diet, the differences, in fact, were not significant. In general, there were fewer sex differences in the bile acid composition of the liver and no significant differences between the two dietary regimens. The bile acid composition of the plasma was not significantly different from that of the intestinal tract, except that no sex differences in the distribution of CDCA or h y o D O CA were found. As in the case of liver, there were no differences between the two dietary regimens. DISCUSSION
This study has demonstrated that the size of the bile acid pool in Wistar rats supplied by High Oak Ranch (Toronto), weighing 200 + 25 g, and maintained on Purina Chow diet is 40-60 gmoles/100 g body wt. This value is at least twice that of published estimates and closely approximates that calculated by Shefer et al. (9) to be required for inhibition of bile acid synthesis under basal conditions. As reported previously, the bulk of the bile acid pool is located in tile intestinal tract (1), and its size and composition are affected by diet (12). In the studies reported here, the bile acid pool of animals maintained on a semisynthetic diet was only 60% of that found in animals maintained on standard laboratory chow. The observation that both the concentration and relative composition of the bile acids in the
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studied. Twenty-four animals, 12 male and 12 female, were kept in a constant temperature The bile acid pool was found to be ca. environment (22 C) in darkness from 7 p.m. to 50 /amoles/100 g body wt in male and 7 a.m. and allowed water ad libitum. One week female rats maintained o n standard laborafter admission to the animal facilities, six male atory chow and ca. 30 #moles/100 g and six female animals were started on a b o d y wt in those maintained on a powdered semisynthetic diet (Teklad Mills, standard semisynthetic diet. The distribuTD-72460 Basal Diet: casein 27%, sucrose tion of bile acids within the pool was 63.9%, corn oil 5%, salt mixture USP XIV 4%, similar in plasma, liver, and intestinal and vitamin mixture 0.1%). Three weeks after tract, except for a higher concentration admission to the animal facility and after an of deoxycholic acid in the intestinal tract. 18-hr fast, the animals were sacrificed by Sex differences in bile acid composition exsanguination under ether anesthesia. were found to be influenced by the dietAs much blood as possible was removed via ary regimen of the animals. the aorta, the intestinal tract was removed after ligation of the distal esophagus and rectum, and INTRODUCTION the liver was removed after ligation of the comThe bile acid pool of the rat has been esti- mon bile duct. Plasma was obtained by centrimated by analysis of the bile acids of the intes- fugation of the blood at 3,000 x g for 20 rain at tinal contents (1), of the bile produced after 4 C. The intestinal tract and the liver were each creation of a biliary fistula (2,3), and of the homogenized vigorously in normal saline, 2 portal blood (4). It has also been estimated ml/g tissue, using a close fitting Teflon-glass more indirectly by isotopic means (5,6). The homo genizer. To 5 ml of plasma, and to aliquots of the pool size in these studies has ranged from ca. 12 to 25 gmoles bile acid/100 g body wt (7,8). intestinal tract and liver homogenates, were From studies on the feedback regulation of bile added tracer amounts of cholyl[ 1-14C] glycine acid synthesis in the rat, Shefer et al. (9) have (CA[1-14C]G) (Amersham Searle, Arlington estimated that the pool of cholyl-taurine in Heights, IL, sp act 49.4 mCi/mmole) and a male rats is ca. 35-40 #moles/100 g body wt. known amount of 5/3-cholanic acid (Applied These workers suggested that previous estimates Science Laboratories, Inc., State College, PA). of the size of the bile acid pool were too low Portions of the plasma and tissue homogenates and that this might explain previous failures to were then extracted with 20 volumes of 0.1% demonstrate a regulatory effect of bile acids on a m m o n i u m hydroxide in absolute ethanol for 4 hr at 60 C. The supernatant obtained after cenbile acid biosynthesis (10). Because of the controversial data in the liter- trifugation at 3,000 x Z for 30 rain at 4 C was saved and this extraction procedure repeated ature and because such information was essential for the interpretation of other investi- twice. The three ethanol extracts were comgations to be conducted in our laboratory, we bined and evaporated to dryness under nitrogen undertook a direct analysis of the size of the after aliquots were taken for radioactivity analbile acid pool in the rat. Because this pool can ysis. Studies of the radioactivity of these exvary with the sex (11) and nutritional status tracts showed an average recovery of 90% of (12) of the animal and because our experi- the added CA[ 1-14C] G. The residue was taken mental program involved the use of a well- up in 5N NaOH and subjected to hydrolysis at defined but semisynthetic diet, we studied both 115 C overnight in sealed tubes. The neutral m a l e a n d female rats on both standard lipids were removed by three extractions of the alkali solution with petroleum ether at 30-60 C laboratory chow and the semisynthetic diet. and, after acidification on ice with 2.5N HC1 to pH 1, the acidic steroids were removed by reMETHODS peated extractions with diethyl ether. The Wistar rats obtained from High Oak Ranch diethyl ether extracts were pooled, checked for (Toronto, Canada) and weighing ca. 200 g were r a d i o a c t i v i t y , and then evaporated under nitrogen. Previous studies in our laboratory 1Yousef is a Canadian Hepatic Foundation Scholar. confirmed that these conditions of alkaline ABSTRACT
93
M.M. FISHER, G. KAKIS, AND I.M. YOUSEF
94
TABLE I Distribution of Bile Acid Pool in Wistar Rats a Purina Chow diet Male Female Intestinal tract (umole/lO0 g body wt) Liverb (gmole/g liver) Blood (lumole/100 ml plasma)
Semisynthetic diet Male Female
50.40 +- 5.10
51.6 :~ 5.40
29.30 + 0.60
27.8 -+ 1.0
0.19 ~ 0.01
0.33 :t 0.01
0.18 -+0.01
0.19 + 0.01
2.96 -+ 0.24
2.97 +- 0.22
1.99•
1.97+-0.12
aValues: mean -+standard error; six animals/group. bLiver wt (g): male, 9.3 +- 0.5; Female, 8.1 +- 0.4 (no significant difference). hydrolysis were equally effective for both glycine and taurine conjugated bile acids (1 I). The completeness of the laydrolysis was indicated by the absence of 14C_radioactivity in the diethyl ether extracts and of CA[1-14C]G in the aqueous layer. The bile acids were methylated, and the trifluoroacetate and trimethyl silyl derivatives of the methylated bile acids were analyzed by gas chromatography as previously described (11,13). The final concentrations of bile acid were calculated on the basis of reference to the internal standard of 5/3cholanic acid (11). Student's t test was used for the statistical analysis, with P > 0.05 indicating no significant difference. RESULTS
The bile acid content of the intestinal tract, blood, and liver of the rats in these studies is presented in Table I. It can be calculated from the data that the total bile acid pool was ca. 100 /~moles in those animals on the Purina Chow diet and 60 ~moles in those on the semisynthetic diet. The reduction in pool size associated with the semisynthetic diet was reflected to an equal degree in the blood, liver, and intestinal tracts. It is apparent that 97% of the bile acid pool is located in the intestinal tract, regardless of sex or diet. There was no sex difference in the size of the bile acid pool in these animals, nor in the concentration of bile acids in the intestinal tract or blood. However, the female rats did have a significantly higher concentration of bile acids in the liver (P < 0.02). The bile acid composition of the intestinal tract, blood, and liver is presented in Table II. Cholic acid (CA) comprised 55-73% o f the bile acid pool. In the intestinal tract, the relative concentrations of CA, lithocholic acid (LCA), and, in those receiving the semisynthetic diet, of chenodeoxycholic acid (CDCA) were signifiLIPIDS, VOL. 11, NO. 2
cantly higher (P < 0.02) in the female rats. The relative concentrations of deoxycho/ic acid ( D O C A ) , /3-muricholic acid (/3MCA), and hyodeoxycholic acid (HyoDOCA) were significantly higher (P < 0.05) in the male. Diet did not appear to influence the relative bile acid composition of the intestinal tract of male rats. Although female rats on the laboratory chow diet appeared to have relatively more CA and relatively less CDCA than did female rats on the semisynthetic diet, the differences, in fact, were not significant. In general, there were fewer sex differences in the bile acid composition of the liver and no significant differences between the two dietary regimens. The bile acid composition of the plasma was not significantly different from that of the intestinal tract, except that no sex differences in the distribution of CDCA or h y o D O CA were found. As in the case of liver, there were no differences between the two dietary regimens. DISCUSSION
This study has demonstrated that the size of the bile acid pool in Wistar rats supplied by High Oak Ranch (Toronto), weighing 200 + 25 g, and maintained on Purina Chow diet is 40-60 gmoles/100 g body wt. This value is at least twice that of published estimates and closely approximates that calculated by Shefer et al. (9) to be required for inhibition of bile acid synthesis under basal conditions. As reported previously, the bulk of the bile acid pool is located in tile intestinal tract (1), and its size and composition are affected by diet (12). In the studies reported here, the bile acid pool of animals maintained on a semisynthetic diet was only 60% of that found in animals maintained on standard laboratory chow. The observation that both the concentration and relative composition of the bile acids in the
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