Uptake of Taurocholic Acid in Human Hepatocytes Isolated From Livers of Donors of Different Age

Bile acids are major synthetic products of the liver. Moreover, after biliary secretion and intestinal reabsorption, they are efficiently extracted from the plasma and are re-excreted into bile, which results in enterohepatic cycling. This enterohepatic circulation of bile acids is impaired during human development (1), which places the infant at risk for clinical cholestasis. Suchy and Balistreri (1) hypothesized that the impairment is caused in part by the immaturity of bile acid transport by the liver. They showed that in rat hepatocytes isolated from rats up to 2 month of age (puberty) there is a progressive increase in taurocholic acid uptake with age, but no data on age-related uptake in human liver were available until now. Taurocholic acid, an anionic compound, has been extensively used in transport studies of bile acids. In rat hepatocytes the uptake of taurocholic acid is carrier mediated and approximately 80% sodium dependent at a concentration of 5 to 50 µM (2,3). In human liver, plasma membrane vesicles, and human hepatocytes, uptake of taurocholic acid also appears to be partly sodium dependent (4,5). In earlier studies, a wide variation in uptake rate of taurocholic acid was recorded in human hepatocytes (4), which could be only partly explained by the different adenosine triphosphate(ATP) concentrations in the hepatocytes (6). In the present study we assessed whether there is immaturity of bile acid transport during human development, by using human hepatocytes. We investigated the uptake of taurocholic acid in human hepatocytes in relation to the age of the donors of human liver tissue, categorizing them in two groups, before and after puberty (aged less than 15 years and aged 15 or more years, respectively). MATERIALS AND METHODS The following compounds were obtained from the indicated sources: collagenase P from Boehringer Mannheim (Mannheim, Germany); bovine serum albumin from Organon Teknika (Boxtel, The Netherlands); sodium taurocholate from Fluka (Buchs, Switzerland); Percoll from Pharmacia (Uppsala, Sweden);[3H(G)]taurocholic acid (specific activity 3.5 Ci/mmol; Du Pont-NEN Research Products, Boston, MA, U.S.A.). University of Wisconsin organ preservation solution was from Du Pont Critical Care (Waukegan, IL, U.S.A.). All other chemicals were of analytical grade and were obtained from commercial sources. Human Liver Material Human liver tissue was obtained from livers procured from multiorgan donors (Tx-livers) or from patients after partial hepatectomy because of metastases of colorectal carcinoma (PH-livers). Consent from the legal authorities and from the families concerned was obtained for the explantation of organs for transplantation purposes. In the case of the PH-livers, consent from the patients concerned was obtained for the use of liver tissue for research purposes. The research protocols were approved by the medical ethical committee of our institution. The liver tissue was procured and the human hepatocytes isolated as described by Olinga et al.(7) Uptake Experiments The uptake experiments with 21 µM taurocholic acid in isolated human hepatocytes were performed as described by Sandker et al.(4) The rate constant for uptake (kin) and excretion (kout) was calculated as described earlier(4). Adenosine triphosphate in the cells was determined immediately after isolation and after 30 minutes of incubation under carbogen gassing at 37°C in a shaking water bath, to restore ATP content. The analysis of ATP was performed as described by Olinga et al.(8). RESULTS The uptake of taurocholic acid was linear for 3 minutes and reached a plateau in 30 to 60 minutes. From the time curve of the uptake of taurocholic acid in human hepatocytes, fractional rate constants were calculated and compared with the age of the donors and the patients undergoing a partial hepatectomy (Table 1). The uptake rate constant of taurocholic acid in human hepatocytes from donors younger than 15 years(range, 6 weeks to 14 years) was significantly lower than the group older than 15 years (range, 22-64 years). The ATP concentration in hepatocytes from the group younger than 15 years was not significantly different from that in the group older than 15 years (Table 1). No difference in transport parameters or ATP content was observed between cells from adults donors or patients undergoing a partial hepatectomy(7).TABLE 1: Fractional rate constant for uptake (kin[10-3/min]) and excretion (kout [10-3/min]) of 21µM taurocholic acid and the ATP concentration (in nmol/106 cells) after 30 minutes in human hepatocytes from different age groupsDISCUSSION Large variations were found in the initial taurocholic acid uptake rate in human hepatocytes, as reported earlier (4,7,9). We previously showed a significant correlation between ATP content and the fractional uptake rate of taurocholic acid, indicating ATP-dependent uptake of taurocholic acid. Because the ATP content of the human hepatocytes seems variable, the variation in taurocholic acid uptake rate observed in the present study could be explained in part by ATP dependency. However, in the present study the fractional rate constants observed in cells from humans younger than 15 years (before puberty) did not correlate with the ATP content of the hepatocytes. Furthermore, in these cells, a significantly lower fractional rate constant for uptake was recorded, regardless of the ATP content in the cells of these young children. Similar differences in uptake of taurocholic acid were noted after 24 hours of culturing these cells from the same livers (F. Kuipers, personal communication). This indicates that uptake differences are maintained during culture and are not an artifact of the experimental setup used in this study. No differences were recorded in the rate constant for excretion(kout) between the two age groups. Thus we concluded that the uptake rate of taurocholic acid was not only dependent on ATP content but also on the age of the donor. Interestingly, Suchy and Balistreri (1) reported that in rat hepatocytes from developing rats (until puberty), no difference in affinity for the carrier was observed during maturation, but that the number of binding sites or transport sites was reduced compared with the number in mature rats. Moreover, posttranslational protein modification of the carrier may still be insufficient in developing rats (10). The current results may indicate that the uptake rate of bile acids in childhood until puberty is lower than in adults. In infancy, a relatively low biliary secretion of bile acids and elevated concentration of bile acids in serum is observed in humans (11), which could be explained by a still immature uptake system for bile acids or by an underdeveloped excretion system for the bile acids. On the basis of our results, we concluded that a lower uptake of bile acids was responsible for the lower bile secretion rate in infancy. Whether this was caused by a lower density of uptake carriers in the sinusoidal membrane for bile acids or to a lower affinity of bile acids for the carrier cannot be differentiated yet. As a consequence of decreased bile secretion, biliary elimination of many drugs and organic anions such as bilirubin may be impaired. In humans in infancy, bile acids are predominantly conjugated with taurine, in contrast to adults, whose major bile acid derivative is glycine conjugated (12). Split liver and reduced-size liver transplantations are used to transplant donor organs of adults into children. Taking into account the results presented here, the question arises of what may be the physiologic influence of the relatively high hepatic uptake rate and the different conjugation pattern of bile acids of adult livers transplanted in these children. In conclusion, uptake but not excretion of taurocholic acid in human hepatocytes seems age dependent-that is, if groups of donors aged less than 15 years and those aged more than 15 years are compared. This difference in uptake rate may partly explain the low biliary output and the elevated concentrations of bile acids in serum in infants, as reported by others(11). To elucidate whether the density of bile acid uptake carriers in the hepatocyte plasma membranes in infancy is lower compared with that in adults, experiments are underway to quantitate the putative uptake carrier for bile acids immunohistochemically(13). Acknowledgment: The study was supported by grants from the Alternatives to Animal Experiments Platform, Rÿswÿk, The Netherlands; Organon International BV, Oss, The Netherlands; and Solvay Duphar BV, Weesp, The Netherlands. The experiments were performed in co-operation with the Human Liver Group Groningen.