Volume 136, Issue 2, Pages 673-682.e1 (February 2009) Effective Treatment of Unconjugated Hyperbilirubinemia With Oral Bile Salts in Gunn Rats  Frans J.C. Cuperus, Anja M. Hafkamp, Rick Havinga, Libor Vitek, Jaroslav Zelenka, Claudio Tiribelli, J. Donald Ostrow, Henkjan J. Verkade  Gastroenterology  Volume 136, Issue 2, Pages 673-682.e1 (February 2009) DOI: 10.1053/j.gastro.2008.10.082 Copyright © 2009 AGA Institute Terms and Conditions

Figure 1 UDCA or CA administration decreases plasma UCB concentration in Gunn rats. Gunn rats (n = 6 per group) were fed the control diet for 6 weeks, followed by: dietary UDCA or CA supplementation in doses that were increased every 2 weeks for 10 weeks (A); dietary UDCA or CA supplementation (0.5 wt% each), or no supplementation for 8 days (B); or dietary UDCA or CA supplementation (0.5 wt% each), or no supplementation for 6 weeks (C). Plasma UCB values at T0 (μmol/L) in B: controls, 241 ± 22; UDCA, 260 ± 25; CA, 251 ± 26 (not significant). Data represent mean ± SD. *P < .05; **P < .01; ***P < .001, compared with plasma bilirubin at T0 (A), or compared with controls (B and C). The used doses of 0.05 wt%, 0.1 wt%, 0.5 wt%, 1.0 wt%, and 1.5 wt% corresponded with a dietary bile salt intake (mg/24 h/kg BW) of, respectively, 32 ± 2, 62 ± 5, 317 ± 23, 633 ± 50, and 948 ± 73. Gastroenterology 2009 136, 673-682.e1DOI: (10.1053/j.gastro.2008.10.082) Copyright © 2009 AGA Institute Terms and Conditions

Figure 2 Short-term UDCA or CA administration to Gunn rats increases fecal bile salt excretion (A); changes the composition of bile salts excreted via the feces (B); increases biliary bile salt excretion (C); and changes the composition of bile salts excreted via the bile (D). Gunn rats (n = 6 per group) were fed the control diet for 6 weeks, followed by dietary UDCA or CA supplementation (0.5 wt% each), or no supplementation for 8 days. Feces were collected during a 4-day period before (pretreatment period) and after (treatment period) dietary randomization. At 8 days, bile was collected during 30 minutes. Data represent mean ± SD. *P < .01; **P < .001; †P < .05. Statistical analysis in feces: 4-day pretreatment period (area under the curve) versus 4-day treatment period (area under the curve). Statistical analysis in bile: UDCA or CA versus controls. LC, lithocholic acid; M, muricholic acid; DC, deoxycholic acid; C, cholic acid; CDC, chenodeoxycholic acid; HDC, hyodeoxycholic acid; UDC, ursodeoxycholic acid; HC, hyocholic acid. Gastroenterology 2009 136, 673-682.e1DOI: (10.1053/j.gastro.2008.10.082) Copyright © 2009 AGA Institute Terms and Conditions

Figure 3 Short-term UDCA or CA administration to Gunn rats increases fecal urobilinoid excretion (A); increases fecal UCB excretion (B); increases fecal urobilinoid + UCB excretion (C); and does not affect biliary urobilinoid + UCB excretion (D). For experimental setup, please refer to Figure 2. Data represent mean ± SD. †P = .06; *P < .05; **P < .01. Statistical analysis in feces: 4-day pretreatment period (area under the curve) versus 4-day treatment period (area under the curve). Statistical analysis in bile: UDCA or CA versus controls. Gastroenterology 2009 136, 673-682.e1DOI: (10.1053/j.gastro.2008.10.082) Copyright © 2009 AGA Institute Terms and Conditions

Figure 4 Bilirubin pool sizes and plasma UCB concentrations in Gunn rats were positively correlated. Gunn rats (n = 6 per group) were fed the control diet for 6 weeks, followed by dietary UDCA or CA supplementation (0.5 wt% each), or no supplementation for 6 weeks. After 6 weeks of treatment, tracer 3H-UCB was intravenously administered to determine UCB kinetics. Plasma was collected every 12 hours over 60 hours. Specific activity of plasma bilirubin declined semilogarithmically with time in each group. Gastroenterology 2009 136, 673-682.e1DOI: (10.1053/j.gastro.2008.10.082) Copyright © 2009 AGA Institute Terms and Conditions

Figure 5 Fractional biliary and transmucosal fluxes of UCB and UCB derivatives in Gunn rats, 6 weeks after randomization. Fractional UCB fluxes are expressed as %, and fractional UCB-derivative fluxes are expressed as equivalent% of the bilirubin pool size that is excreted per hour. [a] fractional turnover of UCB; [b] fractional biliary UCB excretion; [c] fractional biliary UCB-derivative excretion; [d] fractional fecal excretion of UCB + UCB derivatives; [e] estimated net transmucosal flux of UCB from the blood into the intestinal lumen, calculated as [a] – [b]. The magnitude of flux [d] equals that of [a] in a steady-state, assuming that UCB turnover equals the fecal excretion of UCB + UCB derivatives. EHC, enterohepatic circulation. Ovals in upper left of each panel show bilirubin pool size (μmol/100 g BW; mean ± SD). For calculation of fractional fluxes, please refer to Methods section. Gastroenterology 2009 136, 673-682.e1DOI: (10.1053/j.gastro.2008.10.082) Copyright © 2009 AGA Institute Terms and Conditions

Supplemental Figure 1 Short-term UDCA or CA administration to Gunn rats changes biliary bile salt composition. For experimental setup, please refer to Figure 2. Data represent mean ± SD. †P<0.05–P<0.001, compared with controls. Gastroenterology 2009 136, 673-682.e1DOI: (10.1053/j.gastro.2008.10.082) Copyright © 2009 AGA Institute Terms and Conditions