Chenodeoxycholic Acid Induced Liver Injury in Pregnant and Neonatal Baboons CHARLES K. McSHERRY, M.D., KEVIN P. MORRISSEY, M.D., RICHARD L. SWARM, M.D., PATRICIA S. MAY, B.A., WENDELL H. NIEMANN, D.V.M., FRANK GLENN, M.D.
The prolonged feeding of chenodeoxycholic acid produces From the Department of Surgery, hepatic injury in both pregnant and non-pregnant baboons. The New York Hospital-Cornell Medical Center CDC feeding does not adversely affect ovarian function 525 East 68 Street, and no teratogenic effects of this bile acid were noted in New York, N.Y. 10021 16 live birth and two stillborn progeny of CDC fed animals. However, 10 of the 16 live birth neonates and one stillborn had focal hepatic lesions histologically similar to those observed in the adult animals. In addition one neonate had gross hepatic necrosis. The severity of the liver damage was related to is anticipated in approximately one-third of the patients the content of lithocholic acid in the bile of both the neonates when this compound is discontinued.20 Transient elevaand their mothers. Experiments with 14C-chenodeoxycholic and tions of serum transaminase and other hepatic enzyme "4C-lithocholic acid demonstrate that the lithocholate in the levels are noted in patients treated with CDC. Although enterohepatic circulation of the neonate is derived from the CDC some patients receiving CDC have had liver biopsies fed to the pregnant adult. In the gallbladder bile of the neonate most, but not all, of the lithocholate is conjugated but un- interpreted as normal,3 an accurate assessment of the sulfated. Both newborn and adult baboons sulfate lithocholic acid hepatotoxicity of this compound must await an analysis but to an extent less than that reported for man. Less efficient of sequential liver biopsies in a larger group of patients. sulfation of lithocholic acid in the baboon may exaggerate the The limitations of the efficacy of CDC therapy toxicity of CDC feeding in this species compared to man. Never- gest that prevention rather than treatment should besugthe theless, the potential for adverse effects on the fetal liver to reduce the incidence and goal the of morbidity this must be recognized as a risk associated with the use of chenodisease. In this respect, individuals at increased risk of deoxycholic acid in women of child-bearing age. gallstone formation might benefit most from efforts to
decrease the lithogenicity of bile. Furthermore, small and newly formed cholesterol stones are more susceptible to dissolution than larger stones or stones with a high proportion of calcium or other minerals in their matrix in addition to cholesterol.2 There is clinical as well as biochemical evidence of increased susceptibility to gallstone formation in women taking oral contraceptives.4 Many clinicians also believe that pregnancy is a causative factor in gallstone disease and the experience at The New York Hospital-Cornell Medical Center supports this observation. In the age range below 25 years, more than Presented at the Annual Meeting of the American Surgical Associa- 95% of the patients with cholelithiasis are female and many of these women relate the onset of their symptoms tion, New Orleans, Louisiana, April 7-9, 1976. Supported by National Institutes of Health Grant No. ROI-AM- to pregnancy, especially during the second and third 16287. trimesters.9 CHOLESTEROL GALLSTONES have been dissolved in
some humans by the prolonged oral administration of the primary bile acid, chenodeoxycholic (CDC).21 The mechanism of action is attributed to a decreased hepatic secretion of cholesterol into bile' as well as an expansion of the bile salt pool. Both factors contribute to an increase in the capacity of bile to solubilize cholesterol. Recent reports indicate that the chemical dissolution of gallstones may require the administration of CDC for periods of up to 2 years and that recurrence of calculi
490
Vol. 184 . No. 4
CHENODEOXYCHOLIC ACID INDUCED LIVER INJURY
The following study was performed in baboons to identify the potential hazards of CDC administration during pregnancy to the adult female as well as to the developing fetus. Baboons were selected for this study because they spontaneously form cholesterol gallstones, have a bile composition and enterohepatic circulation similar to man12 and, of the non-human primates, most closely resemble man in cholesterol metabolism.8 Materials and Methods Animal selection and maintenance. Twenty-six adult female baboons (Papio anubis and Papio cynocephalus) weighing between 10 and 16 kg were maintained at The Laboratory for Experimental Medicine and Surgery in Primates (LEMSIP), Tuxedo, N.Y. The animals were caged singly and fed standard Purina monkey chow. After a 45-day quarantine period, all animals had a complete blood count, urinalysis, clinical chemistries (sequential multiple analyzer with 12 determinations (SMA-12)), tuberculin test, and assessment of cage behavior and reproductive function. Laparotomy was performed and the fundus of the gallbladder sutured to the adjacent anterior abdominal wall to permit percutaneous aspiration of gallbladder bile for long-term studies of bile composition and kinetics without reoperation or peroral intubation. Wedge and needle liver biopsies were also obtained. On the basis of identification numbers, 8 animals were assigned to a control group and 18 to an experimental group. Upon completion of baseline studies, all animals were mated during the periovulatory period for 3 to 5 consecutive days with one of two males until pregnant. The experimental animals were fed CDC dissolved in ethanol (0.5 mg/ml). This solution was injected into oranges which were fed to each animal prior to their daily ration of monkey chow. Control animals were fed an equivalent volume of the carrier alone (ethyl alcohol in an orange). The CDC was purchased from Weddel Pharmaceuticals, Ltd., London, and purity of lots was checked by gas-liquid chromatography. All lots were greater than 99. 1% pure. Two baboons were given CDC 18 mg/kg/day for 27 months; 10 were progressed by 2 mg/kg/week from 18 to 38 mg/kg/day and have been maintained at this dosage for periods ranging from 16 to 31 months; 6 animals were started and maintained on 38 mg/ kg/day for periods of 13 to 23 months. The animals were observed daily for general appearance, cage behavior, reproductive function, food consumption or wastage and nature of excreta. They were weighed at monthly intervals. Ketamine hydrochloride and/or phencyclidine hydrochloride were used for procedures requiring sedation and for preoperative anesthesia, respectively. Tissue morphology. Percutaneous liver biopsies were obtained at intervals of 1 to 3 months from all adult
491
control and CDC fed animals. The CDC induced focal histologic damage in the liver was graded on a scale of 1 + to 4+ based upon the extent of focal periportal acute and chronic inflammatory cell infiltrate, bile duct reduplication, extension of the lesions into adjacent parenchyma with confluence between some individual portal areas, and increase in connective tissue in and adjacent to some periportal areas. The progeny of both control and experimental animals were either sacrificed at birth or at intervals up to 18 months of age. Both stillborn and live birth neonates had complete postmortem examinations including skeletal radiographs in selected animals. Five neonates born to control animals were sacrificed at birth and another 5 at 6 months of age. Of the neonates born to CDC fed animals, 7 were sacrificed at birth, two at age 6 months, 5 between the ages of 6 to 12 months, and one at age 18 months. One neonate in this group, age 7 months, has not yet been sacrificed. Neonates not sacrificed at birth were operated upon within the first 3 days of life and at intervals of 1 to 2 months thereafter to obtain needle biopsies of the liver and samples of gallbladder bile. The degree of liver injury in the neonates born to CDC fed animals was classified as minimal, moderate or severe. A minimal hepatic lesion denotes the presence of focal periportal bile duct reduplication and infiltration of inflammatory cells. Moderate hepatic damage signifies wider, more extensive focal periportal inflammatory cell infiltrate and abundant foci of bile duct reduplication. Severe liver damage indicates gross hepatic necrosis in addition to the above microscopic ab-
normalities. Biochemical parameters studied. All adult animals had SMA-12 determinations at monthly intervals for the duration of these experiments. The lipid (cholesterol, phospholipid and bile salt) composition and per cent of individual bile acids (cholic, chenodeoxycholic, deoxycholic and lithocholic) in gallbladder bile were measured in all animals before CDC feeding and at intervals of 1 to 3 months thereafter. The pool size and kinetics (halflife and synthesis rate) of the primary bile acids (cholic and chenodeoxycholic) were determined in 6 control and 10 experimental animals before and at 4 to 6 month intervals after the initiation -qf CDC feeding. Tracer bile acids ('4C-cholic and 14C-chenodeoxycholic) were obtained from New England Nuclear Corporation and purified by thin-layer chromatography. The methods employed for these determinations have previously been
reported.7"2
Studies of maternal and fetal bile acid metabolism. The metabolism of chenodeoxycholic acid and its principal metabolite, lithocholic acid, was studied in adult pregnant and non-pregnant animals as well as newborn baboons. Gallbladder bile was obtained from all of the neonates
492
MCSHERRY AND OTHERS
and their mothers within 72 hours of birth. In the neonates the bile was obtained at autopsy, or, in those animals selected for long-term studies, by laparotomy. The per cent of individual bile acids in maternal and neonatal gallbladder bile was compared. In those neonates born to CDC fed adults and not sacrificed at birth, the per cent distribution of biliary bile acids was restudied at intervals of 1 to 2 months until normal. The following study was performed in one animal to establish the source of lithocholic acid in neonatal bile and to study the transplacental transfer of both chenodeoxycholic and lithocholic acids. Unlabeled chenodeoxycholate (20 mg/kg/day) plus 0.17 ,uCi/kg/day of 14Cchenodeoxycholate was fed to this baboon for 7 months prior to conception and continued during gestation (183 days). At term the animal had a Cesarean section and bile was aspirated from the gallbladder. The neonate was operated upon shortly after birth and a sample of gallbladder bile obtained. The bile samples from both the neonate and its mother were analyzed in an identical manner. One-tenth milliliter of bile was dissolved in 1 ml of ethanol and samples were first solvolyzed with dimethoxypropane and HCI, and then hydrolyzed with 2N NaOH. Neutral steroids were removed at the alkaline pH with petroleum ether; the pH was then adjusted to 1-2, and the bile acids extracted with ethyl acetate. Following methylation, the bile acid extracts and suitable standards were plated on 500 mu Silica Gel G plates using the solvent system iso-octane:ethyl acetate:acetic acid (5:5:1, v/v) to separate the individual bile acids. After drying, the TLC plates were covered with unexposed radiographic film and secured in an x-ray cassette for 2 weeks. The radiographs were then developed and the radioactive standards and the radioactivity in the respective bands identified on the TLC plates. Using the radiographs as guides, the individual bile acid bands on the chromatograph were removed from the plate. The bile acids were eluted from the silica with methanol and dried. The residue was dissolved in 2 ml of methanol, and one-half of this sample was used for measuring radioactivity (dpm) and the other half used to determine the mass of the bile acids by GLC using an OV-210 column. For the latter, trifluoroacetate derivatives were prepared. The capacity of the adult baboon to conjugate and sulfate 14C-lithocholic acid was studied in 2 non-pregnant animals by the method of Palmer.16 10 AuCi[24-14C] lithocholate (specific activity 41 mCi per mmole) was injected intravenously and samples of gallbladder bile aspirated at 2, 8, and 24 hours. One-tenth milliliter of bile from each sample was diluted 1:10 with methanol, vortexed and the supernatant removed. One-tenth milliliter of the supernatant plus suitable standards were then plated on 500 mu Silica Gel G plates using the solvent
Ann. Surg.
o
October 1976
system n-butanol:tris:propionic acid (50:9.25:0.75:v/v) to separate the individual bile acids. After overnight drying, the TLC plates were covered with unexposed radiographic film and secured in x-ray cassettes for 1 week. The developed radiographs and the standards, identified by phosphomolybdic acid on the TLC plates, were used to identify and remove the sulfated and nonsulfated conjugates of lithocholate separately from the TLC plates with Hursh funnels. The bile acids were eluted from the silica with hot methanol and dried under N2. The radioactivity of each of the samples was determined by liquid scintillation counting and the ratio of sulfated and conjugated lithocholate to conjugated lithocholate alone was determined by the ratio of the counts. An additional study was performed to measure the capacity of the liver of the newborn baboon to conjugate and sulfate lithocholic acid. Within minutes of delivery, a newborn baboon was given 3 ,uCil4C-lithocholic acid. A sample of gallbladder bile from this baboon was obtained by laparotomy at age 3 days. The per cent of sulfated and unsulfated conjugates of lithocholic acid were measured in this bile sample by the method described above. To determine if the conjugated lithocholate perfusing the liver of the developing fetus was sulfated or unsulfated, the folldiking experiment was performed. A pregnant near-term baboon was given 70 ,uCi 14Clithocholic acid intravenously and the neonate delivered by Cesarean section 6 days later. Three days after delivery, samples of gallbladder bile were obtained from this adult baboon and-its neonate at the same time. In both bile samples the `r cent sulfated and unsulfated conjugated lithochola were determined.
-Results Animal health and reproduction. All animals, control and experimental, remained healthy throughout the study. Exclusive of anticipated changes during pregnancy, both groups of animals increased their mean weight by 1.5 kg. There were no adverse gastrointestinal signs such as vomiting or diarrhea in the CDC fed animals. In the time span of this study, all 8 control and 15 of the 18 (83.3%) CDC fed animals have had at least one pregnancy. Menstrual cycles were regular in all of the animals including those that failed to become pregnant. Control animals had a total of 14 pregnancies and CDC fed animals, 20. There were two spontaneous abortions and two stillbirths in both the control and experimental groups. Thus it appears that chenodeoxycholic acid does not adversely affect ovarian function. Tissue morphology. In the adult control animals, the initial as well as sequential liver biopsies obtained at intervals of 3 months over the 31 month period of study were essentially normal. The current assessment of the de-
Vo.184QA klA No. 4
V-1
CHENODEOXYCHOLIC ACID INDUCED LIVER INJURY
A
A~ ~ ~~
- -
~
~
~
~
~
~
~
493 ~
~~.~(
A
-A
a.7K4
X.
Z,.",~
**~ ~ ..
.-
*..', ~
-
~
X
~ ~ ~~
~
444
~
~
~
~
.~~
~~~~~~~~~~~~~~~
~
~
~
~
~
4
"4.~~~~~~~~~~~~~~;S
d.
1-
W ~~
~
~
~
~
ON.~~~~~~~~~~~~~~~~~~~.
4~h4
~
64',.
~~~~~~~~~~~~#~~~~~' ,~ ~ ~.~ _ -
~~~~~~~~~~--~~~~~~~~~)F' -
4 --4.~~~~4~-
--"'--7----
~
~
~
~ -
~
~
~
~
~ ~
-.
-
.
'4~~~~~~~~~~~~~~~~~~~8
~~~4 ~ ~ ~~
1.4W
5
--
=
~~'
4~WxN..''4 ~~ t'34.44~~~~~N *
*a.
FIG. 1. Photomicrograph of liver biopsy in CDC fed adult baboon that failed to become pregnant (H & E x96). The extent of focal periportal inflammatory cell infiltration, bile duct reduplication and increased connective tissue was graded 3+ (see text). The histologic features of CDC induced liver injury are similar in those baboons that achieved pregnancy. FIG. 2. The normal portal triad in a neonate baboon born to a control animal (H & E X96). FIG. 3. Focal periportal inflammatory cells and bile duct reduplication in the liver biopsy of a neonate baboon with minimal hepatic injury
(H & E x 153).
FIG. 4. Photomicrograph (H & E x60) of the liver biopsy in a neonate with moderate hepatic damage. The lesions are more extensive and involve
FIG. 5. The cut surface of the liver in the neonate with severe liver injury reveals necrosis and obstruction of a large bile duct. FIG. 6. The same neonate at Fig. 5 (H & E x96). In the area of necrosis there is bile duct transformation of hepatocytes.
494 4+
S
3+_
0@
ADULT HEPATIC 2+_ LESIONS
0
I+_
*
0
0 00
S
O_
NEGATIVE
00 S...0
MINIMAL MODERATE
0
SEVERE
NEONATAL HEPATIC LESIONS FIG. 7. The relationship of the severity of the hepatic lesions in the neonate baboons to the extent of liver injury in their mothers. One adult animal with no evidence of hepatic toxicity gave birth to a neonate with minimal liver injury.
gree
Ann. Surg.
MCSHERRY AND OTHERS
of liver damage in the 15 CDC fed animals that became
pregnant during the course of this study is as follows: one is negative, two have 2+, seven have 3+, and five
9
October 1976
serial liver biopsies at intervals of 1 to 2 months disclosed gradual disappearance of the inflammatory cells and bile duct reduplication such that the biopsies were interpreted as negative at ages 5, 8 and 12 months respectively. One neonate with moderate hepatic damage at birth and followed for 7 months does not yet have histologic evidence of regression of the lesion. Biochemical effects. The per cent distribution of the individual bile acids in bile and the pool size of cholic and chenodeoxycholic acid during the control period and at the conclusion of the study are depicted in Fig. 8. Compared to controls, both pregnant and non-pregnant CDC fed animals have exhibited a marked expansion of the chenodeoxycholic acid pool size and an increase in the per cent of this bile acid in bile. Lithocholic acid, produced by bacterial 7a-dehydroxylation of chenodeoxycholic acid in the lower gastrointestinal tract, also has increased significantly from a mean of 2.0 + 1.5% in controls to 10.4 + 5.4% in the CDC fed animals that became pregnant and to 6% in the three animals that failed to achieve pregnancy. Table 1 depicts the cholesterol, phospholipid, and bile salt concentrations in gallbladder bile in control and experimental animals. CDC feeding resulted in a significant (P < 0.05) decrease in the cholesterol concentration of bile, similar to the effect reported by Adler et al. in humans fed this bile acid. Serum from control animals revealed elevations above
have 4+ lesions. The three other experimental animals that failed to become pregnant have 3+ hepatic lesions. The most recent liver biopsy of one of these animals is depicted in Fig. 1. The histologic characteristics of the focal liver lesions in pregnant and nonpregnant baboons fed CDC did not differ significantly. The degree of liver damage appears to be related to the dose and duration of CDC feeding.13 In two of the three CDC fed animals with hepatic lesions graded as negative or 2+, feeding records in- CDC L CH D dicate that one-eighth to one-third of the CDC impregnated orange was often found in the daily food wastage. This obserCONTROLS (8) r vation was infrequent in animals with hepatic lesions of greater severity. Animals that rejected portions of the I80CDC containing oranges developed hepatic lesions at a X BILE rate slower than animals that vigorously ingested the ACID
EXPERIMENTAL PREGNANT (15) NOTPREGNANT(3) I
oranges.
60h Postmortem studies of the stillborn and live birth neonates of both control and CDC fed animals revealed no osseous or extra-hepatic soft tissue lesions; no terato40[ genic effects of CDC were identified. All of the neonates born to control animals as well as 6 of the 16 live birth neonates and one of the two stillbirths born to experimental 20p animals had no gross or microscopic evidence of liver damage (Fig. 2). Focal hepatic lesions were present in 10 live birth neonates and one stillborn among the progeny of CDC fed baboons. The degree of liver SIZE 181 302 939 172 985 88 injury was classified in the neonates as minimal POOL(mgm) in 4 (Fig. 3), moderate in 5 (Fig. 4) and severe in 56 119 one (Figs. 5 and 6). The severity of the liver lesions in FIG. 8. The per cent distribution of the individual bile acids in bile and the neonates did not necessarily correlate with degree of the pool size of cholic and chenodeoxycholic acid in control animals and in CDC fed experimental baboons. CDC = chenodeoxycholic; hepatic injury in their mothers (Fig. 7). L = lithocholic; CH = cholic; D = deoxycholic acid; (n) = number In three neonates with minimal hepatic lesions at birth, of animals. 541
109
VOl. 184 . NO. 4
CHENODEOXYCHOLIC ACID INDUCED LIVER INJURY
495
TABLE 1. Lipid Composition of Gallbladder Bile in Adult Control and CDC Fed Baboons Experimental
Controls
Cholesterol* Phospholipid Bile salts *
22.6 ± 5.5 38.1 + 15.1 314.0 + 67.8
Amounts expressed in ,umol/ml
t n.s.
= not
Pregnant
+
16.0 33.8 266.3
+ + +
(P value)
Non-pregnant
(P value)
The prolonged feeding of chenodeoxycholic acid produces From the Department of Surgery, hepatic injury in both pregnant and non-pregnant baboons. The New York Hospital-Cornell Medical Center CDC feeding does not adversely affect ovarian function 525 East 68 Street, and no teratogenic effects of this bile acid were noted in New York, N.Y. 10021 16 live birth and two stillborn progeny of CDC fed animals. However, 10 of the 16 live birth neonates and one stillborn had focal hepatic lesions histologically similar to those observed in the adult animals. In addition one neonate had gross hepatic necrosis. The severity of the liver damage was related to is anticipated in approximately one-third of the patients the content of lithocholic acid in the bile of both the neonates when this compound is discontinued.20 Transient elevaand their mothers. Experiments with 14C-chenodeoxycholic and tions of serum transaminase and other hepatic enzyme "4C-lithocholic acid demonstrate that the lithocholate in the levels are noted in patients treated with CDC. Although enterohepatic circulation of the neonate is derived from the CDC some patients receiving CDC have had liver biopsies fed to the pregnant adult. In the gallbladder bile of the neonate most, but not all, of the lithocholate is conjugated but un- interpreted as normal,3 an accurate assessment of the sulfated. Both newborn and adult baboons sulfate lithocholic acid hepatotoxicity of this compound must await an analysis but to an extent less than that reported for man. Less efficient of sequential liver biopsies in a larger group of patients. sulfation of lithocholic acid in the baboon may exaggerate the The limitations of the efficacy of CDC therapy toxicity of CDC feeding in this species compared to man. Never- gest that prevention rather than treatment should besugthe theless, the potential for adverse effects on the fetal liver to reduce the incidence and goal the of morbidity this must be recognized as a risk associated with the use of chenodisease. In this respect, individuals at increased risk of deoxycholic acid in women of child-bearing age. gallstone formation might benefit most from efforts to
decrease the lithogenicity of bile. Furthermore, small and newly formed cholesterol stones are more susceptible to dissolution than larger stones or stones with a high proportion of calcium or other minerals in their matrix in addition to cholesterol.2 There is clinical as well as biochemical evidence of increased susceptibility to gallstone formation in women taking oral contraceptives.4 Many clinicians also believe that pregnancy is a causative factor in gallstone disease and the experience at The New York Hospital-Cornell Medical Center supports this observation. In the age range below 25 years, more than Presented at the Annual Meeting of the American Surgical Associa- 95% of the patients with cholelithiasis are female and many of these women relate the onset of their symptoms tion, New Orleans, Louisiana, April 7-9, 1976. Supported by National Institutes of Health Grant No. ROI-AM- to pregnancy, especially during the second and third 16287. trimesters.9 CHOLESTEROL GALLSTONES have been dissolved in
some humans by the prolonged oral administration of the primary bile acid, chenodeoxycholic (CDC).21 The mechanism of action is attributed to a decreased hepatic secretion of cholesterol into bile' as well as an expansion of the bile salt pool. Both factors contribute to an increase in the capacity of bile to solubilize cholesterol. Recent reports indicate that the chemical dissolution of gallstones may require the administration of CDC for periods of up to 2 years and that recurrence of calculi
490
Vol. 184 . No. 4
CHENODEOXYCHOLIC ACID INDUCED LIVER INJURY
The following study was performed in baboons to identify the potential hazards of CDC administration during pregnancy to the adult female as well as to the developing fetus. Baboons were selected for this study because they spontaneously form cholesterol gallstones, have a bile composition and enterohepatic circulation similar to man12 and, of the non-human primates, most closely resemble man in cholesterol metabolism.8 Materials and Methods Animal selection and maintenance. Twenty-six adult female baboons (Papio anubis and Papio cynocephalus) weighing between 10 and 16 kg were maintained at The Laboratory for Experimental Medicine and Surgery in Primates (LEMSIP), Tuxedo, N.Y. The animals were caged singly and fed standard Purina monkey chow. After a 45-day quarantine period, all animals had a complete blood count, urinalysis, clinical chemistries (sequential multiple analyzer with 12 determinations (SMA-12)), tuberculin test, and assessment of cage behavior and reproductive function. Laparotomy was performed and the fundus of the gallbladder sutured to the adjacent anterior abdominal wall to permit percutaneous aspiration of gallbladder bile for long-term studies of bile composition and kinetics without reoperation or peroral intubation. Wedge and needle liver biopsies were also obtained. On the basis of identification numbers, 8 animals were assigned to a control group and 18 to an experimental group. Upon completion of baseline studies, all animals were mated during the periovulatory period for 3 to 5 consecutive days with one of two males until pregnant. The experimental animals were fed CDC dissolved in ethanol (0.5 mg/ml). This solution was injected into oranges which were fed to each animal prior to their daily ration of monkey chow. Control animals were fed an equivalent volume of the carrier alone (ethyl alcohol in an orange). The CDC was purchased from Weddel Pharmaceuticals, Ltd., London, and purity of lots was checked by gas-liquid chromatography. All lots were greater than 99. 1% pure. Two baboons were given CDC 18 mg/kg/day for 27 months; 10 were progressed by 2 mg/kg/week from 18 to 38 mg/kg/day and have been maintained at this dosage for periods ranging from 16 to 31 months; 6 animals were started and maintained on 38 mg/ kg/day for periods of 13 to 23 months. The animals were observed daily for general appearance, cage behavior, reproductive function, food consumption or wastage and nature of excreta. They were weighed at monthly intervals. Ketamine hydrochloride and/or phencyclidine hydrochloride were used for procedures requiring sedation and for preoperative anesthesia, respectively. Tissue morphology. Percutaneous liver biopsies were obtained at intervals of 1 to 3 months from all adult
491
control and CDC fed animals. The CDC induced focal histologic damage in the liver was graded on a scale of 1 + to 4+ based upon the extent of focal periportal acute and chronic inflammatory cell infiltrate, bile duct reduplication, extension of the lesions into adjacent parenchyma with confluence between some individual portal areas, and increase in connective tissue in and adjacent to some periportal areas. The progeny of both control and experimental animals were either sacrificed at birth or at intervals up to 18 months of age. Both stillborn and live birth neonates had complete postmortem examinations including skeletal radiographs in selected animals. Five neonates born to control animals were sacrificed at birth and another 5 at 6 months of age. Of the neonates born to CDC fed animals, 7 were sacrificed at birth, two at age 6 months, 5 between the ages of 6 to 12 months, and one at age 18 months. One neonate in this group, age 7 months, has not yet been sacrificed. Neonates not sacrificed at birth were operated upon within the first 3 days of life and at intervals of 1 to 2 months thereafter to obtain needle biopsies of the liver and samples of gallbladder bile. The degree of liver injury in the neonates born to CDC fed animals was classified as minimal, moderate or severe. A minimal hepatic lesion denotes the presence of focal periportal bile duct reduplication and infiltration of inflammatory cells. Moderate hepatic damage signifies wider, more extensive focal periportal inflammatory cell infiltrate and abundant foci of bile duct reduplication. Severe liver damage indicates gross hepatic necrosis in addition to the above microscopic ab-
normalities. Biochemical parameters studied. All adult animals had SMA-12 determinations at monthly intervals for the duration of these experiments. The lipid (cholesterol, phospholipid and bile salt) composition and per cent of individual bile acids (cholic, chenodeoxycholic, deoxycholic and lithocholic) in gallbladder bile were measured in all animals before CDC feeding and at intervals of 1 to 3 months thereafter. The pool size and kinetics (halflife and synthesis rate) of the primary bile acids (cholic and chenodeoxycholic) were determined in 6 control and 10 experimental animals before and at 4 to 6 month intervals after the initiation -qf CDC feeding. Tracer bile acids ('4C-cholic and 14C-chenodeoxycholic) were obtained from New England Nuclear Corporation and purified by thin-layer chromatography. The methods employed for these determinations have previously been
reported.7"2
Studies of maternal and fetal bile acid metabolism. The metabolism of chenodeoxycholic acid and its principal metabolite, lithocholic acid, was studied in adult pregnant and non-pregnant animals as well as newborn baboons. Gallbladder bile was obtained from all of the neonates
492
MCSHERRY AND OTHERS
and their mothers within 72 hours of birth. In the neonates the bile was obtained at autopsy, or, in those animals selected for long-term studies, by laparotomy. The per cent of individual bile acids in maternal and neonatal gallbladder bile was compared. In those neonates born to CDC fed adults and not sacrificed at birth, the per cent distribution of biliary bile acids was restudied at intervals of 1 to 2 months until normal. The following study was performed in one animal to establish the source of lithocholic acid in neonatal bile and to study the transplacental transfer of both chenodeoxycholic and lithocholic acids. Unlabeled chenodeoxycholate (20 mg/kg/day) plus 0.17 ,uCi/kg/day of 14Cchenodeoxycholate was fed to this baboon for 7 months prior to conception and continued during gestation (183 days). At term the animal had a Cesarean section and bile was aspirated from the gallbladder. The neonate was operated upon shortly after birth and a sample of gallbladder bile obtained. The bile samples from both the neonate and its mother were analyzed in an identical manner. One-tenth milliliter of bile was dissolved in 1 ml of ethanol and samples were first solvolyzed with dimethoxypropane and HCI, and then hydrolyzed with 2N NaOH. Neutral steroids were removed at the alkaline pH with petroleum ether; the pH was then adjusted to 1-2, and the bile acids extracted with ethyl acetate. Following methylation, the bile acid extracts and suitable standards were plated on 500 mu Silica Gel G plates using the solvent system iso-octane:ethyl acetate:acetic acid (5:5:1, v/v) to separate the individual bile acids. After drying, the TLC plates were covered with unexposed radiographic film and secured in an x-ray cassette for 2 weeks. The radiographs were then developed and the radioactive standards and the radioactivity in the respective bands identified on the TLC plates. Using the radiographs as guides, the individual bile acid bands on the chromatograph were removed from the plate. The bile acids were eluted from the silica with methanol and dried. The residue was dissolved in 2 ml of methanol, and one-half of this sample was used for measuring radioactivity (dpm) and the other half used to determine the mass of the bile acids by GLC using an OV-210 column. For the latter, trifluoroacetate derivatives were prepared. The capacity of the adult baboon to conjugate and sulfate 14C-lithocholic acid was studied in 2 non-pregnant animals by the method of Palmer.16 10 AuCi[24-14C] lithocholate (specific activity 41 mCi per mmole) was injected intravenously and samples of gallbladder bile aspirated at 2, 8, and 24 hours. One-tenth milliliter of bile from each sample was diluted 1:10 with methanol, vortexed and the supernatant removed. One-tenth milliliter of the supernatant plus suitable standards were then plated on 500 mu Silica Gel G plates using the solvent
Ann. Surg.
o
October 1976
system n-butanol:tris:propionic acid (50:9.25:0.75:v/v) to separate the individual bile acids. After overnight drying, the TLC plates were covered with unexposed radiographic film and secured in x-ray cassettes for 1 week. The developed radiographs and the standards, identified by phosphomolybdic acid on the TLC plates, were used to identify and remove the sulfated and nonsulfated conjugates of lithocholate separately from the TLC plates with Hursh funnels. The bile acids were eluted from the silica with hot methanol and dried under N2. The radioactivity of each of the samples was determined by liquid scintillation counting and the ratio of sulfated and conjugated lithocholate to conjugated lithocholate alone was determined by the ratio of the counts. An additional study was performed to measure the capacity of the liver of the newborn baboon to conjugate and sulfate lithocholic acid. Within minutes of delivery, a newborn baboon was given 3 ,uCil4C-lithocholic acid. A sample of gallbladder bile from this baboon was obtained by laparotomy at age 3 days. The per cent of sulfated and unsulfated conjugates of lithocholic acid were measured in this bile sample by the method described above. To determine if the conjugated lithocholate perfusing the liver of the developing fetus was sulfated or unsulfated, the folldiking experiment was performed. A pregnant near-term baboon was given 70 ,uCi 14Clithocholic acid intravenously and the neonate delivered by Cesarean section 6 days later. Three days after delivery, samples of gallbladder bile were obtained from this adult baboon and-its neonate at the same time. In both bile samples the `r cent sulfated and unsulfated conjugated lithochola were determined.
-Results Animal health and reproduction. All animals, control and experimental, remained healthy throughout the study. Exclusive of anticipated changes during pregnancy, both groups of animals increased their mean weight by 1.5 kg. There were no adverse gastrointestinal signs such as vomiting or diarrhea in the CDC fed animals. In the time span of this study, all 8 control and 15 of the 18 (83.3%) CDC fed animals have had at least one pregnancy. Menstrual cycles were regular in all of the animals including those that failed to become pregnant. Control animals had a total of 14 pregnancies and CDC fed animals, 20. There were two spontaneous abortions and two stillbirths in both the control and experimental groups. Thus it appears that chenodeoxycholic acid does not adversely affect ovarian function. Tissue morphology. In the adult control animals, the initial as well as sequential liver biopsies obtained at intervals of 3 months over the 31 month period of study were essentially normal. The current assessment of the de-
Vo.184QA klA No. 4
V-1
CHENODEOXYCHOLIC ACID INDUCED LIVER INJURY
A
A~ ~ ~~
- -
~
~
~
~
~
~
~
493 ~
~~.~(
A
-A
a.7K4
X.
Z,.",~
**~ ~ ..
.-
*..', ~
-
~
X
~ ~ ~~
~
444
~
~
~
~
.~~
~~~~~~~~~~~~~~~
~
~
~
~
~
4
"4.~~~~~~~~~~~~~~;S
d.
1-
W ~~
~
~
~
~
ON.~~~~~~~~~~~~~~~~~~~.
4~h4
~
64',.
~~~~~~~~~~~~#~~~~~' ,~ ~ ~.~ _ -
~~~~~~~~~~--~~~~~~~~~)F' -
4 --4.~~~~4~-
--"'--7----
~
~
~
~ -
~
~
~
~
~ ~
-.
-
.
'4~~~~~~~~~~~~~~~~~~~8
~~~4 ~ ~ ~~
1.4W
5
--
=
~~'
4~WxN..''4 ~~ t'34.44~~~~~N *
*a.
FIG. 1. Photomicrograph of liver biopsy in CDC fed adult baboon that failed to become pregnant (H & E x96). The extent of focal periportal inflammatory cell infiltration, bile duct reduplication and increased connective tissue was graded 3+ (see text). The histologic features of CDC induced liver injury are similar in those baboons that achieved pregnancy. FIG. 2. The normal portal triad in a neonate baboon born to a control animal (H & E X96). FIG. 3. Focal periportal inflammatory cells and bile duct reduplication in the liver biopsy of a neonate baboon with minimal hepatic injury
(H & E x 153).
FIG. 4. Photomicrograph (H & E x60) of the liver biopsy in a neonate with moderate hepatic damage. The lesions are more extensive and involve
FIG. 5. The cut surface of the liver in the neonate with severe liver injury reveals necrosis and obstruction of a large bile duct. FIG. 6. The same neonate at Fig. 5 (H & E x96). In the area of necrosis there is bile duct transformation of hepatocytes.
494 4+
S
3+_
0@
ADULT HEPATIC 2+_ LESIONS
0
I+_
*
0
0 00
S
O_
NEGATIVE
00 S...0
MINIMAL MODERATE
0
SEVERE
NEONATAL HEPATIC LESIONS FIG. 7. The relationship of the severity of the hepatic lesions in the neonate baboons to the extent of liver injury in their mothers. One adult animal with no evidence of hepatic toxicity gave birth to a neonate with minimal liver injury.
gree
Ann. Surg.
MCSHERRY AND OTHERS
of liver damage in the 15 CDC fed animals that became
pregnant during the course of this study is as follows: one is negative, two have 2+, seven have 3+, and five
9
October 1976
serial liver biopsies at intervals of 1 to 2 months disclosed gradual disappearance of the inflammatory cells and bile duct reduplication such that the biopsies were interpreted as negative at ages 5, 8 and 12 months respectively. One neonate with moderate hepatic damage at birth and followed for 7 months does not yet have histologic evidence of regression of the lesion. Biochemical effects. The per cent distribution of the individual bile acids in bile and the pool size of cholic and chenodeoxycholic acid during the control period and at the conclusion of the study are depicted in Fig. 8. Compared to controls, both pregnant and non-pregnant CDC fed animals have exhibited a marked expansion of the chenodeoxycholic acid pool size and an increase in the per cent of this bile acid in bile. Lithocholic acid, produced by bacterial 7a-dehydroxylation of chenodeoxycholic acid in the lower gastrointestinal tract, also has increased significantly from a mean of 2.0 + 1.5% in controls to 10.4 + 5.4% in the CDC fed animals that became pregnant and to 6% in the three animals that failed to achieve pregnancy. Table 1 depicts the cholesterol, phospholipid, and bile salt concentrations in gallbladder bile in control and experimental animals. CDC feeding resulted in a significant (P < 0.05) decrease in the cholesterol concentration of bile, similar to the effect reported by Adler et al. in humans fed this bile acid. Serum from control animals revealed elevations above
have 4+ lesions. The three other experimental animals that failed to become pregnant have 3+ hepatic lesions. The most recent liver biopsy of one of these animals is depicted in Fig. 1. The histologic characteristics of the focal liver lesions in pregnant and nonpregnant baboons fed CDC did not differ significantly. The degree of liver damage appears to be related to the dose and duration of CDC feeding.13 In two of the three CDC fed animals with hepatic lesions graded as negative or 2+, feeding records in- CDC L CH D dicate that one-eighth to one-third of the CDC impregnated orange was often found in the daily food wastage. This obserCONTROLS (8) r vation was infrequent in animals with hepatic lesions of greater severity. Animals that rejected portions of the I80CDC containing oranges developed hepatic lesions at a X BILE rate slower than animals that vigorously ingested the ACID
EXPERIMENTAL PREGNANT (15) NOTPREGNANT(3) I
oranges.
60h Postmortem studies of the stillborn and live birth neonates of both control and CDC fed animals revealed no osseous or extra-hepatic soft tissue lesions; no terato40[ genic effects of CDC were identified. All of the neonates born to control animals as well as 6 of the 16 live birth neonates and one of the two stillbirths born to experimental 20p animals had no gross or microscopic evidence of liver damage (Fig. 2). Focal hepatic lesions were present in 10 live birth neonates and one stillborn among the progeny of CDC fed baboons. The degree of liver SIZE 181 302 939 172 985 88 injury was classified in the neonates as minimal POOL(mgm) in 4 (Fig. 3), moderate in 5 (Fig. 4) and severe in 56 119 one (Figs. 5 and 6). The severity of the liver lesions in FIG. 8. The per cent distribution of the individual bile acids in bile and the neonates did not necessarily correlate with degree of the pool size of cholic and chenodeoxycholic acid in control animals and in CDC fed experimental baboons. CDC = chenodeoxycholic; hepatic injury in their mothers (Fig. 7). L = lithocholic; CH = cholic; D = deoxycholic acid; (n) = number In three neonates with minimal hepatic lesions at birth, of animals. 541
109
VOl. 184 . NO. 4
CHENODEOXYCHOLIC ACID INDUCED LIVER INJURY
495
TABLE 1. Lipid Composition of Gallbladder Bile in Adult Control and CDC Fed Baboons Experimental
Controls
Cholesterol* Phospholipid Bile salts *
22.6 ± 5.5 38.1 + 15.1 314.0 + 67.8
Amounts expressed in ,umol/ml
t n.s.
= not
Pregnant
+
16.0 33.8 266.3
+ + +
(P value)
Non-pregnant
(P value)
