Apple Computer, Inc., and Biosoft (Cambridge, UK). as compared with the control group, i.e., animals whose mothers were not cholestatic (Fig. 1A). Liver weight.
Reversible Impairment of Neonatal Hepatobiliary Function by Maternal Cholestasis MARIA J. MONTE,1 ANA I. MORALES,1 MIGUEL AREVALO,2 INMACULADA ALVARO,1 ROCIO I. R. MACIAS,1 1 AND JOSE J. G. MARIN
The effect of total blockage of maternal biliary excretion during the last third of the pregnancy on the maturation of hepatobiliary function was investigated in neonatal rats. Extrahepatic obstruction of the common bile duct on day 14 of pregnancy induced a marked enhancement in serum bilirubin—mainly conjugated bilirubin— and bile acid concentrations as compared with shamoperated pregnant rats. Excretion of bile acids by the kidney was significantly increased, whereas fecal elimination of these compounds was almost abolished. Most of the cholestatic mothers (CMs) (77%) were able to carry pregnancy to term and lactation until weaning (21 days after birth). The body and liver weights of their offspring were lower than for offspring of control healthy mothers in all postnatal periods considered. Serum bile acid concentrations were higher in the fetuses and neonates of CMs. This difference was evident up to 1 week after weaning and disappeared in young adult animals (8 weeks old). When the bile secretion rate was investigated in these animals at 4 or 8 weeks of age, no significant difference was found as far as nonstimulated bile flow and bile acid output was concerned. However, the biliary response to stepwise sodium taurocholate (TC) intravenous infusion showed that 4-week-old neonates of CMs had impaired bile acid secretion. Moreover, the maximal secretion rate (SRmax ) for TC was significantly reduced (030%), whereas the choleretic ability of taurocholate was not modified. This alteration was not selective for bile acids. The SRmax for bromosulfophthalein (BSP) was also significantly lowered (040%). These dysfunctions were overcome during subsequent development. No impaired biliary response to either TC or BSP infusion was observed at 8 weeks of age. Morphological abnormalities in the canaliculi were found in animals with impaired biliary function. In summary, these results indicate that maternal cholestasis may profoundly but transiently impair the normal liver maturation. The
Abbreviations: TC, sodium taurocholate; BSP, bromosulfophthalein; CM, cholestatic mothers; SRmax , maximal secretion rate. From the 1Department of Physiology and Pharmacology, and 2Human Anatomy and Histology, University of Salamanca, Spain. Received January 30, 1995; accepted December 4, 1995. Supported in part by the Fondo de Investigaciones Sanitarias de la Seguridad Social (FIS, grant 93/0419), Spain. Address reprint requests to: Jose Juan Garcia Marin, Ph.D., Departamento de Fisiologia y Farmacologia, Campus Miguel de Unamuno, EID-S09, 37007 Salamanca, Spain. Copyright q 1996 by the American Association for the Study of Liver Diseases. 0270-9139/96/2305-0040$3.00/0
importance of the implications derived from these findings both in the nutrition and management of human neonates demands further evaluation of the hepatobiliary function of babies born after alterations of fetal-maternal bile acid homeostasis, such as in maternal obstetric cholestasis. (HEPATOLOGY 1996;23:1208-1217.)
Normal fetal development requires efficient transfer across the placenta. Nutrients are supplied by the mother while certain potentially toxic compounds cross the placenta, mainly in the fetus-to-mother direction. Among the latter are bile acids. Both in humans and experimental animals, the onset of bile acid biosynthesis precedes the development of efficient mechanisms involved in enterohepatic circulation.1 Hepatic insufficiency with respect to bile acid secretion is found in both premature and full-term infants.2 This is accompanied by a marked immaturity of small intestinal transport of bile acids.3-5 Because the accumulation of bile acids within the fetus can seriously challenge the viability of pregnancy, such compounds must be transferred to the mother, whose liver is able to excrete them into bile. Therefore, fetal-maternal bile acid homeostasis requires both functional placental transfer and normal maternal hepatobiliary function. Obstetric cholestasis is a pathological situation characterized by a 10- to 100-fold increase in postprandial bile acid concentrations in the mother and, occasionally, jaundice. This disease is more common than previously suspected and occurs during the second half or the third trimester of the pregnancy in women who were healthy before and after the pregnancy.6 Besides skin pruritus, subclinical fat malabsorption, and mildly abnormal results of routine biochemical liver tests, deleterious effects on the mother are absent. By contrast, cholestasis of the pregnancy, as well as any other situation leading to marked increases in maternal serum bile acid concentrations, may seriously impair the placental clearance of fetal bile acids, leading to a dangerous accumulation of these compounds within the fetus. Owing to the organotropism of bile acids, the highest risk of being harmed should be expected for the fetal liver. Indeed, pathological increases in bile acid liver contents induced by extrahepatic obstruction in the adult rat have been associated with marked alterations in liver metabolism.7-9 Bile acid toxicity may also play an important role in the prematurity, low birth weight,
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intrapartum asphyxia, and increased fetal mortality that have been reported to occur with an enhanced incidence during severe obstetric cholestasis.10-12 Moreover, the teratogenicity of administered secondary bile acids has been shown to influence embryonic rat development both in vitro13 and in vivo.14 The sensitivity to the deleterious effects of administered secondary bile acids is not restricted to the period of early differentiation but is extended to the period of organogenesis.15 Taken together, the above-mentioned situations suggest that the maturation of liver function can be challenged by alterations in the fetal-maternal bile acid homeostasis. Using a laboratory animal model, we investigated how total blockage of maternal biliary excretion during the last third of the pregnancy and the subsequent accumulation of endogenous bile acids in the mother-fetus tandem affect the development of hepatobiliary function as investigated by quantitation of the biliary response to the exogenous administration of sodium taurocholate (TC) and bromosulfophthalein (BSP) to infant (4-week-old) and young (8-week-old) adult rats. MATERIALS AND METHODS Chemicals. TC (more than 95% pure by thin-layer chromatography), BSP, 3a-hydroxysteroid dehydrogenase, diaphorase, and resazurin were purchased from Sigma Chemical Co. (St. Louis, MO). All other chemicals were from Merck (Darmstadt, Germany). Animals. Nonfasting pregnant Wistar CF rats (approximately 250 g) (Faculty of Pharmacy, Salamanca, Spain) and their offspring were used. They were fed on commercial pelleted rat food (Panlab, Madrid, Spain) and water ad libitum. Lighting was controlled by a timer that permitted light between 8 AM and 8 PM. All animals received humane care as outlined in ‘‘Guide for the Care and Use of Laboratory Animals’’ (NIH Publication no. 80-23, revised 1985). Experimental cholestasis was induced surgically by complete extrahepatic obstruction on day 14 of pregnancy. Under ether anesthesia, a median laparotomy was performed. Approximately 0.5 cm of the upper portion of the common bile duct was dissected free from the surrounding structures. Using a nonabsorbable suture, a double ligation separated by approximately 2 mm was performed. The common bile duct was divided between the ligations. The abdominal incision was then closed. On day 14 of pregnancy, the sham-operated controls underwent laparotomy and manipulation of the liver and small intestine before the incision was closed. All operations were performed under sterile conditions. Rats were allowed to recover from anesthesia in a warmed cabinet. They were then housed in individual metabolic cages permitting separate urine and feces collection until the end of the pregnancy. After birth, the neonates were regularly weighed and some of them killed to obtain blood samples. The litters were weaned on day 21 after birth. In some cases, a cesarean section was performed on day 20 of pregnancy, and the fetuses were killed to obtain liver and blood samples. Maternal blood samples were collected from the carotid artery to determine serum bilirubin and total bile acid concentrations. Experimental Protocol. The experiments on bile formation were performed at 10 AM on days 28 or 56 after birth. Surgery and bile collection were performed under sodium pentobarbital anesthesia (50 mg/kg body weight, intraperitoneally,
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Nembutal N.R., Abbott, Madrid, Spain). Tracheotomy followed by a median laparotomy were performed. Cannulation of the common bile duct and jugular vein was performed as described previously.16 Rectal temperature was maintained at 37 { 0.57C throughout the experiments. After an equilibration period of 15 minutes, bile was collected in preweighed vials at 10-minute intervals. After two basal bile samples, a stepwise infusion of either TC or BSP was performed through the jugular vein. Doses were the following: 0.3, 0.6, 0.9, 1.2, 1.5, 1.8, 2.1, 2.4, 2.7, 3.0, 3.3, and 3.6 mmol/min for TC and 0.3, 0.6, 0.9, 1.2, 1.5, 1.8, and 2.1 mmol/min for BSP (10 minutes each). At the end of the experimental period, the animals were killed by exsanguination. Livers were removed and weighed. Analytical Methods. Bile flow was determined gravimetrically. The concentration of 3a-hydroxy bile acids in bile was measured enzymatically using 3a-hydroxysteroid dehydrogenase.17 To measure serum bile acid concentrations, liquidsolid extraction of serum samples was performed.18 Serum samples were diluted (1:4, vol/vol) with 0.4 mol/L NH4HCO3 and heated to 647C for 2 hours. Samples were centrifuged for 15 minutes at 5,000 g. The resulting supernatants were heated again for 10 minutes and extracted using reversephase octadecylsilane-bonded silica cartridges (Sep-Pak, Waters-Millipore, Madrid, Spain). After washing the cartridges with water (10 mL), acetone (3 mL 10% in water), and water (10 mL), bile acids were recovered with 4 mL of methanol. This solution was filtered using a ‘‘Sample Clarification Kit’’ (Waters-Millipore) and dried. To perform total bile acid measurement, the sample was redissolved with 200 mL of methanol. This procedure allows both an efficient elimination of serum components other than bile acids and also increases the concentration of bile acids in the final solution by approximately 20-fold, which is required to perform subsequent analytical procedures. Bile acids in urine were extracted by a modification of the method of Alme´ et al.,19 using a combination of Amberlite XAD-7 (Teknokroma, Barcelona, Spain) and Sep-Pak cartridges. Bile acids in feces were extracted by the method of Setchell et al.20 After extraction, total 3a-hydroxybile acid concentrations in serum, urine, and feces were determined by stoichiometric conversion of 3a-hydroxy-bile acid into 3-keto-bile acid together with the generation of a fluorescent molecule of resorfin from one of resazurin by the sequential action of 3a-hydroxysteroid dehydrogenase and diaphorase in the presence of the oxidized form of nicotinamide-adenine dinucleotide as cofactor.17,21 Both total and conjugated plasma bilirubin were measured by an adaptation of the diazo method of Jendrassik and Grof22 for total bilirubin. BSP was determined spectrophotometrically at 580 nm after dilution of the bile with 0.1 N NaOH. Histological Studies. Rats were anesthetized with sodium pentobarbital, and the livers were excised and cut into approximately 2-mm3 fragments. The samples were fixed by immersion in 2.5% glutaraldehyde in 0.2 mol/L sodium cacodylate buffer, pH 7.4. After washing in cacodylate buffer, the pieces were postfixed in 1% osmium tetroxide in 0.2 mol/L cacodylate buffer, dehydrated in graded ethanol, embedded in gelatin capsules with Durcupan resin, and polymerized in an oven at 607C for 48 hours. Semithin sections were cut, stained with toluidine blue, and observed by light microscopy. Ultrathin sections were cut, mounted on grids, stained with uranyl acetate and lead citrate, and examined with a Zeiss EM 900 transmission electron microscope (Zeiss, Oberkochen, Germany) at 60 kV. Statistical Analysis. Results are expressed as means { SE. To calculate the statistical significance of differences, the
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TABLE 1. Effect of Extrahepatic Obstructive Cholestasis on Fecal and Urinary Bile Acid Excretion and on Serum Bile Acid and Bilirubin Concentrations in the Mother Rat 20th Day of Pregnancy
Third Week of Pregnancy
Control Cholestasis
Weaning Day
Feces
Urine
Serum
Bile Acid Excretion (mmol/wk)
Bile Acid Excretion (mmol/wk)
Bile Acid (mmol/L)
Bile Acid (mmol/L)
Total Bilirubin (mmol/L)
Conjugated Bilirubin (mmol/L)
n
261.4 { 54.0 10.2 { 1.2*
3.9 { 0.9 86.2 { 14.9*
13.2 { 5.9 219.7 { 26.7*
14.8 { 1.9 175.3 { 16.0*
2.6 { 0.1 128.0 { 17.1*
1.3 { 0.1 104.1 { 13.9*
5 5
NOTE. Cholestatic rats underwent bile duct ligation on day 14 of pregnancy. Feces and urine samples were collected during the third week of pregnancy. Serum samples were taken 6 days (20th day of pregnancy) or 28 days (weaning day) after surgery. Sham-operated rats were used as controls. Values are means { SE. The results were compared with the control group by the Student’s t test. * P õ .001.
Student’s t test was used. Regression lines were obtained by the least square method. The mean values of y-axis intercepts and slopes were compared by covariance analysis. Statistical analysis was performed on a Macintosh SE computer (Apple Computer, Inc., Cupertino, CA) with programs supplied by Apple Computer, Inc., and Biosoft (Cambridge, UK). RESULTS Maternal Cholestasis. Complete mechanical obstruction of the common bile duct on gestational day 14 in the pregnant rat led to a loss of the major route responsible for the excretion of bile acids in the motherfetus tandem. As a result, fecal bile acid excretion was dramatically (26-fold) reduced (Table 1). Under these circumstances, urinary excretion provides an alternative excretory pathway that tries to maintain bile acid homeostasis. Indeed, during the third week of pregnancy, urinary elimination of 3a-hydroxy-bile acids, which are only a part of the bile acids present in urine, was 21.5-fold higher in cholestatic than in control pregnant rats (Table 1). In spite of this increased excretion, a marked hypercholanemia was observed to occur in cholestatic animals (Table 1). Signs of jaundice were also evident, and weight loss as compared with shamoperated pregnant rats was found during the first week after surgery (data not shown). In spite of maternal cholestasis, 77% of pregnancies were viable, and the rats were able to feed their litters until weaning; i.e., at day 21 after birth. At this time serum bile acid concentrations in cholestatic mothers (CMs) were found to be higher (11-fold) than in control animals (Table 1). Marked hyperbilirubinemia also was observed. This was mainly (81.3%) accounted for by an 80-fold increase in conjugated bilirubin concentrations in maternal serum (Table 1). As a result of bilirubin accumulation in maternal blood, placental transfer of this compound from the fetuses toward the mother was probably impaired; thus, total serum bilirubin concentration as measured in fetuses collected at 20th day of pregnancy showed significantly (P õ .05) higher values in the offspring of cholestatic mothers (CM group, 4.74 { 0.63 mmol/L) than in the control (3.18 { 0.22 mmol/ L) group (both n Å 6, from three different litters).
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Neonatal Hypercholanemia and Nonstimulated Bile Formation. Maternal cholestasis during the last third
of pregnancy and lactation had a marked effect on the weight gain and functional development of the neonates. Body weight was always lower in the CM group as compared with the control group, i.e., animals whose mothers were not cholestatic (Fig. 1A). Liver weight (Table 2) was also significantly lower in the CM group. However, the liver weight to body weight ratio was not significantly different. In the control group, at the 20th day of pregnancy, serum bile acid concentrations in the fetuses were slightly but not significantly higher than in their mothers (15.1 { 4.0 mmol/L vs. 13.2 { 5.9 mmol/L, respectively). However, this value was significantly increased early after birth (27.1 { 5.7 mmol/L, P õ .05 vs. both fetuses and healthy mothers). This neonatal physiological hypercholanemia has also been observed in humans by other authors.2 In agreement with previous studies on rats,23 neonatal hypercholanemia was first reduced 1 week after birth and then increased to reach a maximal value at 4 weeks of age. This was followed by a decrease leading to values of serum bile acid concentrations similar to those of adults by 8 weeks of age. This evolution in serum bile acid concentrations was also observed in newborns of the CM group, except for the following points: (1) Serum bile acid concentrations were 3.5-fold lower in the fetuses than in their mothers. (2) After birth, a significant (P õ .05) decrease in neonatal versus fetal serum bile acid concentrations was observed. (3) Neonatal hypercholanemia was significantly more pronounced than that found in the offspring of healthy mothers (Fig. 1B). This difference between the control and CM groups disappeared in 8week-old rats. On investigating the ontogenesis of bile secretion, other authors24 have found similar basal bile flow and bile acid output at 4 and 8 weeks of age in the rat when the results are expressed per body weight. However, in this study as compared with results obtained at 4 weeks after birth (Table 2), young adult rats (8 weeks old) showed significantly (P õ .01) lower nonstimulated bile flow and bile acid output when
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these parameters are expressed per gram of liver tissue. No significant differences between the control and CM groups were found regarding nonstimulated bile function at either 4 or 8 weeks. Effect of Maternal Cholestasis on Stimulated Bile Formation. To investigate whether higher cholanemia
in the neonates and infants of the CM group than in the control group was caused by impaired liver ability to handle a bile acid load, such as that occurring postprandially, an intravenous stepwise infusion of TC was performed on infant rats. Experiments performed on 4-week-old rats indicated that bile acid output and bile flow stimulated by TC administration were significantly reduced in the CM group compared with those found in control animals (Fig. 2). By contrast, when these experiments were performed later during postnatal development, i.e., on 8-week-old rats, marked difference in the hepatobiliary response to an exogenous load to TC was not found between CM offspring and control animals (Fig. 3). In fact, a slightly higher bile acid output and significantly higher bile flow in CM group were observed. This was most probably attributable to an experimental artifact, because these animals received the same amount of infused TC, but the livers of animals belonging to CM group were smaller (Table 2). At 4 weeks, the value of the maximal secretion rate for TC (SRmax ) was found to be significantly decreased (030%) in the CM group (Fig. 4). In control rats, SRmax was lower (057%) at 8 weeks than at 4 weeks of age. Moreover, in young adult animals, similar SRmax values were found in the control and the CM groups (Fig. 4). The so-called bile acid–independent fraction of bile flow and bile acid choleretic ability were calculated from the relationship between bile acid output and bile flow (Fig. 5). Approximately 7 mL of bile were collected per micromole of secreted bile acid, regardless of the experimental group considered. No effect of maternal cholestasis on bile acid–independent fraction was found. This parameter was similar in all groups. The higher choleresis observed at 8 weeks in the CM group seems to be related to the existence of a slightly higher bile acid output (Fig. 3).
FIG. 1. Effect of maternal cholestasis on (A) body weight and (B) serum bile acid concentrations in the newborn rat. CM group includes offspring of rats that underwent total bile duct ligation on day 14 of pregnancy. Values are means { SE. The number of animals at each point was between 10 and 40, belonging to at least five different litters. The results were compared with the control group by the Student’s t test. *P õ .05.
TABLE 2. Effect of Maternal Cholestasis on Liver Weight, Basal Biliary Function, and Serum Bilirubin Levels in Infant and Young Adult Rats
Time After Birth (wk)
Control CM Control CM
4 4 8 8
Liver Weight (g)
3.22 2.44 9.73 8.63
{ { { {
0.21 0.12* 0.43† 0.51
Liver/Body Weight Ratio (%)
3.87 4.21 4.03 4.31
{ { { {
0.25 0.19 0.12 0.23
Basal Bile Flow (mL/min/g liver)
2.43 2.32 1.88 1.71
{ { { {
0.25 0.13 0.09† 0.33
Serum Bilirubin Basal Bile Acid Output (nmol/min/g liver)
103.6 106.6 64.04 67.87
{ { { {
16.5 8.7 6.6† 8.2
Total (mmol/L)
2.68 5.19 1.86 2.71
{ { { {
0.25 0.35* 0.15† 0.24*
Conjugated (mmol/L)
1.32 2.90 1.01 0.94
{ { { {
0.25 0.23* 0.13 0.20
NOTE. Values are means { SE (n ¢ 8, belonging to at least five different litters). Basal nonstimulated bile flow and bile acid output were calculated from the average of two 10-minute bile samples collected at the beginning of experiments on bile formation. Student’s t test was used to compare the results. * P õ .05 as compared with the results obtained on the group of offspring whose mothers underwent complete extrahepatic biliary obstruction on day 14 of pregnancy (CM) with the control group. † P õ .05 as compared with control groups at 8 and 4 weeks after birth.
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FIG. 2. Time course of (A) bile acid output and (B) bile flow stimulated by stepwise TC infusion in young rats of 4 weeks of age. TC was infused through the jugular vein from minute 20 at the following increasing doses: 0.3, 0.6, 0.9, 1.2, 1.5, 1.8, 2.1, 2.4, 2.7, 3.0, 3.3, and 3.6 mmol/min for 10 minutes each. Bars show the accumulated TCinduced increase in (A) bile acid output and (B) bile flow from minute 20 to minute 140. Values are means { SE. Offspring of cholestatic mothers (CM group, n Å 8) who underwent total bile duct ligation on day 14 of pregnancy were compared with control rats of the same age (control group, n Å 5). *P õ .05; **P õ .01; significantly different by the Student’s t test.
FIG. 3. Time course of (A) bile acid output and (B) bile flow stimulated by stepwise TC infusion in young rats of 8 weeks of age. TC was infused through the jugular vein from minute 20 at the following increasing doses: 0.3, 0.6, 0.9, 1.2, 1.5, 1.8, 2.1, 2.4, 2.7, 3.0, 3.3, and 3.6 mmol/min for 10 minutes each. Bars show the accumulated TCinduced increase in (A) bile acid output and (B) bile flow from minute 20 to minute 140. Values are means { SE. Offspring of cholestatic mothers (CM group, n Å 6) who underwent total bile duct ligation on day 14 of pregnancy were compared with control rats of the same age (control group, n Å 6). *P õ .05; **P õ .01; significantly different by the Student’s t test.
Effect of Maternal Cholestasis on Neonatal BSP Excretion. To elucidate whether the transitory impair-
ment in biliary formation observed in the offspring of cholestatic mothers affected only the mechanisms responsible for bile acid secretion by the liver, the output into bile of a non–bile acid cholephilic organic anion such as BSP was investigated. Figure 6 shows that the biliary excretion of BSP was markedly reduced in the CM group 4 weeks after birth. As was observed for TC, the SRmax for BSP was significantly lower (040%) in the CM animals as compared with the controls (Fig. 7). This impairment in the excretion of organic anions was also transient and disappeared 4 weeks later (Figs. 6 and 7). Morphological Alterations in Offspring of Cholestatic Rats. No relevant modifications in liver samples
obtained from the CM group were observed in histological preparations by light microscopy. However, at electron microscopy, fetuses from the CM group showed differences compared with control animals (Fig. 8). Thus, in some fields, fetuses from CM group showed slightly dilated bile canalicular spaces with an apparent reduction in the number of microvilli (Fig. 9). In other fields, the lumen of bile canaliculi was filled with a finely granular material, and a loss of microvilli also
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FIG. 4. SRmax of bile acids in young rats at 4 weeks or 8 weeks of age. TC was infused through the jugular vein from minute 20 at the following increasing doses: 0.3, 0.6, 0.9, 1.2, 1.5, 1.8, 2.1, 2.4, 2.7, 3.0, 3.3, and 3.6 mmol/min, for 10 minutes each. Values are means { SE from 5 to 8 experiments in each group. Offspring of cholestatic mothers (CM group) that underwent total bile duct ligation on day 14 of pregnancy were compared with control rats of the same age (control group) by the Student’s t test. *P õ .05; †P õ .05; as compared with the control group at 4 weeks.
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FIG. 5. Relationship between bile flow and bile acid output in rats of (A) 4 weeks or (B) 8 weeks of age. TC was infused through the jugular vein from minute 20, at the following increasing doses: 0.3, 0.6, 0.9, 1.2, 1.5, 1.8, 2.1, 2.4, 2.7, 3.0, 3.3, and 3.6 mmol/min for 10 minutes each. Offspring of cholestatic mothers (CM group, n Å 8 experiments at 4 weeks, n Å 6 at 8 weeks) who underwent total bile duct ligation on day 14 of pregnancy were compared with control rats of the same age (control group, n Å 5 experiments at 4 weeks, n Å 6 at 8 weeks).
was seen. In most cases, many vesicular structures were observed around the bile canaliculi of CM group. By contrast, only few of them were seen in most control samples. In some instances, intercellular spaces appeared dilated with disruption of the normal tight and gap junctions. At 4 weeks of age, control animals showed liver parenchymal structure very similar to that observed in adult rats. This maturity was also observed as far as canalicular morphology was concerned (Fig. 10). However, at this age, animals belonging to the CM group showed profound morphological alterations. Among them, enlarged bile canaliculi with apparent loss of microvilli and the presence of multilamellar bodies within the canalicular lumen (Fig. 11). These multilamellar assemblies were also seen in the cytoplasm around canaliculi. Histological studies carried out at 8 weeks of age showed no relevant differences between control and CM groups (data not shown). DISCUSSION
Bile acids cross the placenta mainly in the fetal-tomaternal direction through specific transport systems
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FIG. 6. Time course of BSP bile output in young rats of (A) 4 weeks or (B) 8 weeks of age. BSP was infused through the jugular vein from minute 20 at the following increasing doses: 0.3, 0.6, 0.9, 1.2, 1.5, 1.8, and 2.1 mmol/min for 10 minutes each. Bars show the accumulated BSP total bile output from minute 20 to minute 90, at (A) 4 weeks and (B) 8 weeks. Values are means { SE. Offspring of cholestatic mothers (CM group, n Å 6 for each age) that underwent total bile duct ligation on day 14 of pregnancy were compared with control rats of the same age (control group, n Å 6 for each age). *P õ .05; **P õ .01; ***P õ .001; significantly different by Student’s t test.
FIG. 7. SRmax of BSP in young rats of 4 weeks or 8 weeks of age. BSP was infused through the jugular vein from minute 20, at the following increasing doses: 0.3, 0.6, 0.9, 1.2, 1.5, 1.8, and 2.1 mmol/ min, for 10 minutes each. Values are means { SE from six experiments in each group. Offspring of cholestatic mothers (CM group) that underwent total bile duct ligation on day 14 of pregnancy were compared with control rats of the same age (control group) by Student’s t test. **P õ .01; compared with the control group.
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Although bile acids may be detected in the rat from very early on during fetal life, maturation of the fetal pool of bile acids occurs during the last week of pregnancy, when the size of bile acid pool increases by two orders of magnitude.3 We reasoned that changes affecting the homeostasis of maternal-fetal bile acid during this period may substantially impair the normal maturation of hepatobiliary function. Our results support this hypothesis. This functional alteration is not permanent but is reversed during postnatal development. Moreover, our results indicate that morphological alterations in the developing canaliculi may be among the causes or the consequences of high values of serum bile acid concentrations in the offspring of cholestatic rats. To understand the underlying mechanism of this transient dysfunction, further studies are needed. Only some speculative explanations can now be offered. Marked maternal hypercholanemia seems to be the most likely candidate to account for these effects. However, other possibilities cannot be ruled out. Among them, bilirubin, another potentially toxic compound with marked hepatotropism, might be involved. Experimental evidence obtained both in humans33 and in laboratory animals34,35 suggests an important role for the
FIG. 8. Canalicular structure as observed by transmission electron microscopy. The liver sample was obtained from a control fetus of a rat on the 20th day of pregnancy. BC, bile canaliculus; M, mitochondrion. (Original magnification 22,9601.)
located at both sides of trophoblast plasma membrane.25-28 The reversibility properties of some of these carriers29 support the concept of bile acid transfer also in the mother-to-fetus direction.30 Besides the possibility of direct synthesis by the fetal liver,31 the existence of a probably minor bile acid flux from the maternal blood could account for the presence of secondary bile acids in the fetal bile acid pool, in spite of the absence of intestinal bacterial flora in the fetus. Moreover, a correlation between fetal and maternal serum bile acid concentrations does exist.31 Therefore, it can be reasonably assumed that accumulation of bile acids in the maternal blood by bile duct ligation in the pregnant rat would result in an alteration in the overall transplacental bile acid exchange. This is consistent with twofold higher serum bile acid concentrations at birth in the neonates of cholestatic mothers. In contrast to the situation in the adult, in the fetal rat an important fraction of the bile acid pool is located within the liver. Thus, enhanced exposure of the fetal liver to these compounds can be expected to occur in fetuses of bile duct– ligated rats.3 This does not necessarily mean an increased bile acid pool size. Previous work by other authors32 in which bile duct ligation was performed at the ninth day of pregnancy in the rat reported a 30-fold increase in maternal serum bile acid concentrations together with a reduction in the fetal bile acid pool size.
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FIG. 9. Canalicular morphology as observed by transmission electron microscopy. The liver sample was obtained from a 20-dayold fetal rat belonging to the CM group. The micrograph shows a dilated canaliculus with partial loss of microvilli. BC, bile canaliculus; M, mitochondrion. (Original magnification 22,9601.)
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themselves. In the intestine, both upregulation in pup rats37 and downregulation in the adult rat38 have been described. In the adult, exposure of the liver to a high bile salt flux can increase the SRmax for taurocholate.39,40 Moreover, an adaptative downregulation induced by bile salt depletion has also been reported.41 Therefore, the regulatory effect on liver carriers could partly explain the observed reduction in the ability of the neonatal livers of the CM group to secrete bile acids into bile. The detergent properties of bile acids have been suggested to play an important role in causing hepatic dysfunction after bile duct obstruction.42 This could account for less specific effects such as the observed impairment in the secretion of organic anions that do not share the transport systems with bile acids, such as BSP. Moreover, bile acid accumulation within the hepatobiliary system induces severe modifications in the organization of the gap junctional systems,43 which have been observed in fetal livers of CM group. This could affect intercellular communication and synchronized development of the fetal liver tissue. Furthermore, cholestasis induces profound changes in the tight junctional pattern characterized by an increased number of discontinuities in the strands and the formation of numerous free-ending loops,43 which suggests in-
FIG. 10. Canalicular structure as observed by transmission electron microscopy. The liver sample was obtained from a 4-week-old control rat. BC, bile canaliculus. (Original magnification 22,9601.)
placenta in the clearance of fetal bilirubin toward the maternal blood, from where it is excreted by the liver. Our results suggest that this situation may be modified during complete obstruction of the maternal bile duct. However, probably because of vectorial properties of bilirubin transfer by the placenta, high increase in maternal serum bilirubin concentrations induced much less marked fetal hyperbilirubinemia. The fact that bile acids are almost absent in the maternal intestine implies that the absorption of fat and liposoluble vitamins would be seriously challenged. The effect of the loss of these nutrients on fetal liver maturation is unknown but presumably important. Our results indicate that alterations in neonatal hepatobiliary function are evident even 1 week after weaning, despite the fact that during this period the animals have had direct access to food containing appropriate amounts of fat and vitamins. This means either that nutritional factors are not the only ones responsible for this effect or that 1 week of normal nutrient supply is not enough time to reverse the impairment caused by previous deficiency. Regarding the role of bile acids in this effect, several possibilities can be entertained. Hypercholanemia may have an indirect effect by inducing the secretion of circulating factors.36 The expression of functional bile acid carriers has been reported to be affected by bile acids
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FIG. 11. Canalicular structure as observed by transmission electron microscopy. The liver sample was obtained from a 4-week-old rat belonging to the CM group. Note the evident multilamellar bodies within a bile canaliculus (arrows). (Original magnification 22,9601.)
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creased paracellular permeability. Although disruption of the normal tight junctional complexes in the fetuses of the CM group was observed, it is uncertain whether this was involved in the impaired biliary function observed in infant rats of the CM group, because no change in choleretic ability of secreted bile acids was found. Rather our results are consistent with a reduction of the number of functional canaliculi, caused by the obstruction of some of them by multilamellar bodies invading the canalicular lumen. Changes in cytoskeletal elements leading to lower canalicular contractility cannot be ruled out. Finally, a more profound interaction of bile acids with liver maturation at genetic level should be considered. Bile acids have been shown to be able to modify liver growth during regeneration by inhibiting DNA synthesis.44 If this also occurs during fetal development, it could explain in part the delay in the expression of specific liver functions such as bile formation. High serum bile acid concentrations, occurring in humans and laboratory animal neonates by dispersion of bile acids from the enterohepatic into the systemic circulation, is attributable to passive jejunal bile acid absorption45 together with a relative deficiency in hepatic bile acid clearance.23 Accordingly, the existence of an additional latent dysfunction in bile acid secretion may become particularly evident postprandially and may contribute to further enhancement of serum bile acid concentrations in these neonates. These findings have important clinical implications, because bile acids are required for the solubilization and absorption of essential nonpolar lipids and liposoluble vitamins as well as for optimal activity of the two principal lipases of the infant, namely, human breast milk lipase and pancreatic lipase.46,47 Another interesting consideration as far as human babies are concerned is the importance of neonatal hyperbilirubinemia in this type of patient. If, similarly to what happens for BSP, bilirubin excretion is also impaired in these neonates, this could contribute to enhancing their serum bilirubin concentrations. In summary, these results indicate that complete obstruction of the common bile duct in pregnant rats results in a marked but transient impairment of hepatobiliary function during the postnatal life of their offspring. The importance of the implications derived from these findings both in the nutrition and care of human neonates demands further assessment of the hepatobiliary function of babies born after maternal obstetric cholestasis. Acknowledgment: The authors thank M. I. Hernandez Rodriguez for her secretarial help, M. C. Gonzalez Mesonero for her technical assistance, J. Villoria Terron and J. F. Martin Martin for caring for the animals, and A. Perez and V. Alonso for their excellent work and assistance in electron microscopy studies. Thanks are also due to Nicholas Skinner for his help in the preparation of the manuscript.
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