Clinicaland Experimental Pharmacology & Physiology (1979) 6 , 117-120.
SHORT COMMUNICATION THE HEPATIC TRANSPORT OF SODIUM [ 14C]TAUROCHOLATE IN FOETAL SHEEP R. B. Sewell," K. J. Hardy, R. A. Smallwood and N. E. Hoffmant Gastroenterologv Unit, Departments of Medicine and Surgery, Austin Hospital, Heidelberg, Victoria, Australia (Received 25 May 1978;revision received 5 August 1978)
SUMMARY
1. The biliary excretion of a tracer pulse of sodium [14C]taurocholate injected into the portal vein was compared in adult and near-term foetal sheep. 2. Biliary excretion was virtually complete in both adult and foetus, although appreciably faster in the adult. 3. These results indicate that at birth the mechanisms for bile salt uptake and excretion in the sheep liver are well established. Key words: bile salts, foetus, hepatic transport, placental transfer. INTRODUCTION At birth, the new born liver must take over a number of excretory functions from the placenta. Some of these functions, e.g. the elimination of bilirubin, are clearly defective in the neonate (Bernstein et al., 1969). Efficient hepatic bile salt transport is central to the biliary elimination of many endogenous and exogenous substances, since bile salts play a key role in bile formation (Small, Dowling & Redinger, 1972). Present evidence suggests that hepatic bile salt transport in the foetal dog near term is remarkably mature (Jackson et al., 1971), but that this is not true of the foetal monkey, where hepatic uptake and excretion are less well established (Little et al., 1975). We have developed an experimental model in sheep to study the maturation of bile salt metabolism in the foetus and neonate. This initial study compares the hepatic transport of sodium 14C-labelled taurocholate in the foetus at term with that of the non-pregnant adult sheep. METHODS
Surgical preparation Foetal experiments were carried out in five pregnant sheep, either Merino or Dorset-Poll, *Present address: Liver Unit, King's College Hospital, London. ?Present address: Gastroenterology Division, University of Texas, Medical School, Houston, Texas, USA. Correspondence: Dr R. A. Smallwood, Department of Medicine, Austin Hospital, Heidelberg, Victoria 3084, Australia. 0305-1870/79/0100-0117$02.00 0 1979 Blackwell Scientific Publications
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from 2 to 5 years of age, between day 140 and day 147 of gestation (term). Three nonpregnant adult sheep, weighing 39.440.5 kg, were studied for comparison. The foetal sheep weighed 2.24.3 kg. The animals had been treated with levamisol phosphate t o clear intestinal parasites and had been fed a lucerne-chaff mixture (50:50,v/v) in metabolic cages. The sheep were fasted overnight before operation. Anaesthesia was induced with intravenous thiopentone sodium (I5 mglkg), and an endotracheal tube was passed. During the experiment, anaesthesia was maintained with a halothane/oxygen mixture using intermittent positive pressure respiration. A maternal carotid artery cannula was used to sample blood for pH, PO, and PCO, and these were maintained within normal limits throughout each experiment. All surgery was done using full surgical sterile techniques. In foetal experiments, the maternal gaU bladder was removed and the common bile duct was cannulated (polyethylene cannula, 1.2 mm i.d.) through a midline laparotomy. The uterus was then opened and the foetus delivered onto a small platform. The foetus and umbilical cord were kept warm and moist with warm saline packs. A right subcostal incision was then made in the foetus, acholecystectomy was performed, and the foetal common bile duct was cannulated (polyethylene cannula 0.60 rnm i.d.). In the adult experiments, cholecstectomy and common bile duct cannulation (polyethylene cannula, 1.2 mm i.d.) were performed. In all experiments, bile from the foetus and the adult was collected in 2 min aliquots. The cannula lengths were measured to calculate the dead-space. At the end of each experiment the foetus and adult were killed.
Experimental design Experiments were begun in the anaesthetized sheep after the establishing of steady bile flow and closure of the surgical incisions. Foetal experiments. A pulse of radiolabelled sodium taurocholate (5 .O pCi of sodium [14C]taur~cholate,specific activity 58 mCi/mmol, New England Nuclear) was injected as a tracer dose into the portal vein in the five foetal sheep. In two foetuses, a simultaneous tracer pulse of tritiated sodium taurocholate (10 pCi of sodium [3H] taurocholate, specific activity 3.39 Ci/mmol, New England Nuclear) was injected into the foetal jugular vein. Foetal and maternal bile were collected in aliquots every 2 min for 120 min and bile was counted for radiolabel in each sample. Adult experiments. A tracer pulse of labelled sodium taurocholate (18 pCi of sodium ['HI taurocholate, New England Nuclear) was injected into the portal vein of three nonpregnant adult sheep. Bile was again collected in aliquots every 2 min for 120 min, and counted for radiolabel. Analytical procedures The purity of the labelled bile acids was found by thin layer chromatography (TLC) t o be > 98%.Radioactivity in bile was measured on a three channel Packard Tricarb liquid scintillation counter using Bray's solution as a scintillant. Quench correction was performed by external standardization. Blood gas analysis was performed on a Radiometer BMS 3, Blood Micro System. TLC was done on aliquots of bile run on silica gel G (0.25 mm) plates using a chloroform-methanol-water-acetic acid solvent system (25 :15 :4:2, v/v/v/v) and conjugated bile acids as standards to assess whether radiolabel was recovered in bile primarily as taurocholate.
Foetal hepatic bile salt transport
119
RESULTS The cumulative excretion of radiolabel in bile following portal vein injection of labelled sodium taurocholate is shown in Fig. 1. The results in five foetuses and three non-pregnant adults are compared. Bile flow in both foetal and adult sheep was steady throughout the experiment. The mean bile flow of the five foetuses was 1 1.O k 1.5 I.tl/kg per min, and of the three adults was 13.7 f 2.4 pl/kg per min. In both adult and foetus, nearly all the injected bile salt was recovered in bile as radiolabelled taurocholate within the experimental period (98.6 k 2.7% and 88.1 k 3.2%). However, in the adult, radiolabel appeared in the bile sooner (2 min us 8 min in the foetus), and there was an appreciably faster peak excretion rate (13.1 ? 1.5% dose/min us 3.8 f 0.5% doselmin) which was achieved earlier (4-6 rnin us 16-18 min). Biliary excretion of radiolabel was virtually complete in the adult by 30 min, while in the foetus radiolabel was still slowly accumulating a t 100 min. Two foetuses were injected simultaneously with [ 14C] taurocholate (into the portal vein) and [3H]taurocholate (into the jugular vein). The biliary excretion of [3H] taurocholate took longer (20-22 min us 16-1 8 min) to reach its maximal rate of excretion (3 .O% dose/min s 100.-E
2
80-
T J r
10
20
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I
I
I
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I
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30
40
50
60
70
80
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I 100
Time (min)
Fig. 1. Excretion of radiolabel in bile after a tracer pulse of sodium [ I4C]taurocholate injected via the portal vein in three adult (0) and five foetal (m) sheep with biliary fistulas. The cumulative excretion of radiolabel into adult bile was 98.6% (s.e.m. = 2.7%)at 100 min, compared with 88.1%(s.e.m. = 3.2%) in foetal bile.
us 3.8% doselmin), but the final cumulative excretion at 100 min was similar to [14C] tauro-
cholate. Approximately 1% of the dose of [3H] taurocholate injected into the foetal jugular vein crossed the placenta to appear in the maternal bile. Following portal vein injection, n o radiolabel was detected in maternal bile over the time of the experiment. DISCUSSION
In the present study we have compared the hepatic transport of sodium taurocholate in foetal and adult sheep. The results indicate that the foetal liver can take up and excrete a pulse of taurocholate with an efficiency that approaches that of the adult liver. However, the adult liver excretes the injected tracer dose appreciably faster, suggesting that hepatic bile salt transport in the sheep foetus is not completely mature.
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Foetal hepatic uptake of a tracer dose of [‘*C] taurocholate injected into the portal vein was probably complete at first pass, since no radiolabel crossed the placenta to appear in maternal bile. However, uptake may not have been complete had a larger bolus been used, since evidence in the foetal dog (Smallwood et ul., 1972) suggests that a proportion of the circulating endogenous bile salt pool does escape to the peripheral circulation. Injection of [3H]taur~holateinto the foetal jugular vein did lead to some placental transfer, while injection into the umbilical vein, with first presentation of labelled bile salt to the placenta, results in 30%of the injected dose appearing in maternal bile (R. A. Smallwood, P. Jablonski & J. M. Watts, unpublished results). The fact that placental transfer from mother to foetus can readily occur, suggests the possibility that the placenta may act as an excretory route for bile salts, as it does for other substances such as bilirubin (Bernstein et al., 1969). These results are comparable to those found in the near term foetal dog (Jackson et ul., 1971) and foetal monkey (Little et ul., 1975), in that foetal hepatic uptake and placental transfer of bile salts infused into the foetal peripheral circulation both occur. Of the three species, the foetal monkey appears to have the least well developed hepatic transport mechanism at term. Plasma disappearance of an infused tracer dose of bile salt is comparatively slow, and only 33%of the infused dose is excreted in foetal bile. A similar proportion of the infusate is transferred across the placenta and appears in maternal bile. In contrast, in the foetal dog over 80% of the infused dose appears in foetal bile, and only 1-2% in maternal bile. The present study demonstrates similarly efficient bile salt transport by the foetal sheep liver. It thus appears that hepatic transport of bile salt matures later in the primate, as is the case with the transport of bilirubin. REFERENCES Bemstein, RB., Now, M.J., Piasecki, G.J., Lester, R. & Jackson, B.T. (1969) Bilirubin metabolism in the fetus. Journal of Clinical Investigation, 48, 1678-1688. Jackson, B.T., Smallwood, R.A., Piasecki, G.J., Brown, A.S. Rauschecker, H.F.J. & Lester, R (1971) Fetal bile salt metabolism. I. The metabolism of sodium “C-cholate in the fetal dog. Journal of Clinical Investigation, 50, 1286-1 294. Little, J.M., Smallwood, RA., Lester, R, Piasecki, G.J. & Jackson, B.T. (1975) Bile salt metabolism in the primate fetus. Gastroenterology, 69, 1315-1320. Small, D.M., Dowling, RH. & Redinger, RN. (1972) The enterohepatic circulation of bile salts. Archives of Internal Medicine, 130,552-574. Smallwood, RA., Lester, R., Piasecki, G.J., Klein, P.D.,Greco, R. & Jackson, B.T. (1972) Fetal bile salt metabolism. 11. Hepatic excretion of endogenous bile salt and of a taurocholate load. Journal of Clinical Investigation, 5 1, 1388-1391.
SHORT COMMUNICATION THE HEPATIC TRANSPORT OF SODIUM [ 14C]TAUROCHOLATE IN FOETAL SHEEP R. B. Sewell," K. J. Hardy, R. A. Smallwood and N. E. Hoffmant Gastroenterologv Unit, Departments of Medicine and Surgery, Austin Hospital, Heidelberg, Victoria, Australia (Received 25 May 1978;revision received 5 August 1978)
SUMMARY
1. The biliary excretion of a tracer pulse of sodium [14C]taurocholate injected into the portal vein was compared in adult and near-term foetal sheep. 2. Biliary excretion was virtually complete in both adult and foetus, although appreciably faster in the adult. 3. These results indicate that at birth the mechanisms for bile salt uptake and excretion in the sheep liver are well established. Key words: bile salts, foetus, hepatic transport, placental transfer. INTRODUCTION At birth, the new born liver must take over a number of excretory functions from the placenta. Some of these functions, e.g. the elimination of bilirubin, are clearly defective in the neonate (Bernstein et al., 1969). Efficient hepatic bile salt transport is central to the biliary elimination of many endogenous and exogenous substances, since bile salts play a key role in bile formation (Small, Dowling & Redinger, 1972). Present evidence suggests that hepatic bile salt transport in the foetal dog near term is remarkably mature (Jackson et al., 1971), but that this is not true of the foetal monkey, where hepatic uptake and excretion are less well established (Little et al., 1975). We have developed an experimental model in sheep to study the maturation of bile salt metabolism in the foetus and neonate. This initial study compares the hepatic transport of sodium 14C-labelled taurocholate in the foetus at term with that of the non-pregnant adult sheep. METHODS
Surgical preparation Foetal experiments were carried out in five pregnant sheep, either Merino or Dorset-Poll, *Present address: Liver Unit, King's College Hospital, London. ?Present address: Gastroenterology Division, University of Texas, Medical School, Houston, Texas, USA. Correspondence: Dr R. A. Smallwood, Department of Medicine, Austin Hospital, Heidelberg, Victoria 3084, Australia. 0305-1870/79/0100-0117$02.00 0 1979 Blackwell Scientific Publications
117
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from 2 to 5 years of age, between day 140 and day 147 of gestation (term). Three nonpregnant adult sheep, weighing 39.440.5 kg, were studied for comparison. The foetal sheep weighed 2.24.3 kg. The animals had been treated with levamisol phosphate t o clear intestinal parasites and had been fed a lucerne-chaff mixture (50:50,v/v) in metabolic cages. The sheep were fasted overnight before operation. Anaesthesia was induced with intravenous thiopentone sodium (I5 mglkg), and an endotracheal tube was passed. During the experiment, anaesthesia was maintained with a halothane/oxygen mixture using intermittent positive pressure respiration. A maternal carotid artery cannula was used to sample blood for pH, PO, and PCO, and these were maintained within normal limits throughout each experiment. All surgery was done using full surgical sterile techniques. In foetal experiments, the maternal gaU bladder was removed and the common bile duct was cannulated (polyethylene cannula, 1.2 mm i.d.) through a midline laparotomy. The uterus was then opened and the foetus delivered onto a small platform. The foetus and umbilical cord were kept warm and moist with warm saline packs. A right subcostal incision was then made in the foetus, acholecystectomy was performed, and the foetal common bile duct was cannulated (polyethylene cannula 0.60 rnm i.d.). In the adult experiments, cholecstectomy and common bile duct cannulation (polyethylene cannula, 1.2 mm i.d.) were performed. In all experiments, bile from the foetus and the adult was collected in 2 min aliquots. The cannula lengths were measured to calculate the dead-space. At the end of each experiment the foetus and adult were killed.
Experimental design Experiments were begun in the anaesthetized sheep after the establishing of steady bile flow and closure of the surgical incisions. Foetal experiments. A pulse of radiolabelled sodium taurocholate (5 .O pCi of sodium [14C]taur~cholate,specific activity 58 mCi/mmol, New England Nuclear) was injected as a tracer dose into the portal vein in the five foetal sheep. In two foetuses, a simultaneous tracer pulse of tritiated sodium taurocholate (10 pCi of sodium [3H] taurocholate, specific activity 3.39 Ci/mmol, New England Nuclear) was injected into the foetal jugular vein. Foetal and maternal bile were collected in aliquots every 2 min for 120 min and bile was counted for radiolabel in each sample. Adult experiments. A tracer pulse of labelled sodium taurocholate (18 pCi of sodium ['HI taurocholate, New England Nuclear) was injected into the portal vein of three nonpregnant adult sheep. Bile was again collected in aliquots every 2 min for 120 min, and counted for radiolabel. Analytical procedures The purity of the labelled bile acids was found by thin layer chromatography (TLC) t o be > 98%.Radioactivity in bile was measured on a three channel Packard Tricarb liquid scintillation counter using Bray's solution as a scintillant. Quench correction was performed by external standardization. Blood gas analysis was performed on a Radiometer BMS 3, Blood Micro System. TLC was done on aliquots of bile run on silica gel G (0.25 mm) plates using a chloroform-methanol-water-acetic acid solvent system (25 :15 :4:2, v/v/v/v) and conjugated bile acids as standards to assess whether radiolabel was recovered in bile primarily as taurocholate.
Foetal hepatic bile salt transport
119
RESULTS The cumulative excretion of radiolabel in bile following portal vein injection of labelled sodium taurocholate is shown in Fig. 1. The results in five foetuses and three non-pregnant adults are compared. Bile flow in both foetal and adult sheep was steady throughout the experiment. The mean bile flow of the five foetuses was 1 1.O k 1.5 I.tl/kg per min, and of the three adults was 13.7 f 2.4 pl/kg per min. In both adult and foetus, nearly all the injected bile salt was recovered in bile as radiolabelled taurocholate within the experimental period (98.6 k 2.7% and 88.1 k 3.2%). However, in the adult, radiolabel appeared in the bile sooner (2 min us 8 min in the foetus), and there was an appreciably faster peak excretion rate (13.1 ? 1.5% dose/min us 3.8 f 0.5% doselmin) which was achieved earlier (4-6 rnin us 16-18 min). Biliary excretion of radiolabel was virtually complete in the adult by 30 min, while in the foetus radiolabel was still slowly accumulating a t 100 min. Two foetuses were injected simultaneously with [ 14C] taurocholate (into the portal vein) and [3H]taurocholate (into the jugular vein). The biliary excretion of [3H] taurocholate took longer (20-22 min us 16-1 8 min) to reach its maximal rate of excretion (3 .O% dose/min s 100.-E
2
80-
T J r
10
20
I
I
I
I
I
I
I
30
40
50
60
70
80
90
I 100
Time (min)
Fig. 1. Excretion of radiolabel in bile after a tracer pulse of sodium [ I4C]taurocholate injected via the portal vein in three adult (0) and five foetal (m) sheep with biliary fistulas. The cumulative excretion of radiolabel into adult bile was 98.6% (s.e.m. = 2.7%)at 100 min, compared with 88.1%(s.e.m. = 3.2%) in foetal bile.
us 3.8% doselmin), but the final cumulative excretion at 100 min was similar to [14C] tauro-
cholate. Approximately 1% of the dose of [3H] taurocholate injected into the foetal jugular vein crossed the placenta to appear in the maternal bile. Following portal vein injection, n o radiolabel was detected in maternal bile over the time of the experiment. DISCUSSION
In the present study we have compared the hepatic transport of sodium taurocholate in foetal and adult sheep. The results indicate that the foetal liver can take up and excrete a pulse of taurocholate with an efficiency that approaches that of the adult liver. However, the adult liver excretes the injected tracer dose appreciably faster, suggesting that hepatic bile salt transport in the sheep foetus is not completely mature.
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R . B. Sewell et al.
Foetal hepatic uptake of a tracer dose of [‘*C] taurocholate injected into the portal vein was probably complete at first pass, since no radiolabel crossed the placenta to appear in maternal bile. However, uptake may not have been complete had a larger bolus been used, since evidence in the foetal dog (Smallwood et ul., 1972) suggests that a proportion of the circulating endogenous bile salt pool does escape to the peripheral circulation. Injection of [3H]taur~holateinto the foetal jugular vein did lead to some placental transfer, while injection into the umbilical vein, with first presentation of labelled bile salt to the placenta, results in 30%of the injected dose appearing in maternal bile (R. A. Smallwood, P. Jablonski & J. M. Watts, unpublished results). The fact that placental transfer from mother to foetus can readily occur, suggests the possibility that the placenta may act as an excretory route for bile salts, as it does for other substances such as bilirubin (Bernstein et al., 1969). These results are comparable to those found in the near term foetal dog (Jackson et ul., 1971) and foetal monkey (Little et ul., 1975), in that foetal hepatic uptake and placental transfer of bile salts infused into the foetal peripheral circulation both occur. Of the three species, the foetal monkey appears to have the least well developed hepatic transport mechanism at term. Plasma disappearance of an infused tracer dose of bile salt is comparatively slow, and only 33%of the infused dose is excreted in foetal bile. A similar proportion of the infusate is transferred across the placenta and appears in maternal bile. In contrast, in the foetal dog over 80% of the infused dose appears in foetal bile, and only 1-2% in maternal bile. The present study demonstrates similarly efficient bile salt transport by the foetal sheep liver. It thus appears that hepatic transport of bile salt matures later in the primate, as is the case with the transport of bilirubin. REFERENCES Bemstein, RB., Now, M.J., Piasecki, G.J., Lester, R. & Jackson, B.T. (1969) Bilirubin metabolism in the fetus. Journal of Clinical Investigation, 48, 1678-1688. Jackson, B.T., Smallwood, R.A., Piasecki, G.J., Brown, A.S. Rauschecker, H.F.J. & Lester, R (1971) Fetal bile salt metabolism. I. The metabolism of sodium “C-cholate in the fetal dog. Journal of Clinical Investigation, 50, 1286-1 294. Little, J.M., Smallwood, RA., Lester, R, Piasecki, G.J. & Jackson, B.T. (1975) Bile salt metabolism in the primate fetus. Gastroenterology, 69, 1315-1320. Small, D.M., Dowling, RH. & Redinger, RN. (1972) The enterohepatic circulation of bile salts. Archives of Internal Medicine, 130,552-574. Smallwood, RA., Lester, R., Piasecki, G.J., Klein, P.D.,Greco, R. & Jackson, B.T. (1972) Fetal bile salt metabolism. 11. Hepatic excretion of endogenous bile salt and of a taurocholate load. Journal of Clinical Investigation, 5 1, 1388-1391.
