- Email: [email protected]
Cholate uptake and conjugation by a porcine hepatocyte-based bioartificial liver (BAL)
Al144
AASLD ABSTRACTS
• THE EFFECT OF A NITRIC OXIDE.SYNTHASE INHIBITOR ON SYSTEMIC HAEMODYNAMICS IN CIRRHOTIC PATIENTS D. Patch. PA McCormick. L Greenslade. R Dick. AK Burroughs. UniversityDept of Medici,e. Royal Free, Hospital. London N'W3. U K Aim: Nitric oxide(NO) has been postulated to mediate the hyperdyfiamic circulation in portal hypertension and cirrhosis. We therefore investigated the effect on systemic haemodynamics of a bolus of a NO synthase inhibitor (LNMMA) in cirrhotic patients. Method: 9 patients (Pughs grade B or C) were studied at the time of routine right heart Swann-Ganz catheterisation prior to liver transplantation. Cardiac output and systemic vascular resistance was calculated by standard flow, dilution. A 10mg bolus of L-NMMA was iniected intravenously in all individuals except patient 5 who received 5mg. Cardiac output studies were perforrded 90-180 secs post-dose. Result s were analysed using a student tr test. Results: (given as mean values) CO SVR PVR mmHg DylLCnLSeC "5 Dyn.cm:sec "5 L/min pre ix)st pro post pro post pre ~os{ 108 112 16 17 72n 75{I 14 57 93 9{) 64o 543 35 18 4 6 92 93 827 7 t 5 53 76 93 97 ' 7 8 750 ~27 34 - 36 83 83 9 9 830 772 29 46 83 93 9 9 761 819 49 47 83 93 10 582 611 62 8 82 80 6 4 1164 1224 43 62 9 100 97 3 4 1333 1315 53 54 No significant difference was detected in the change of any parameters (p>0.1). Discussion: L-NMMA at these doses had little effect on systemm or pulmonary haemodynamics in cirrhotic patients No adverse effects were noted. These results suggest that nitric oxide may not be the principle mediator of the vasodilated state of end-stage cirrhosis Further studies with higher doses of L-NMMA are required BP
CHOLATE UPTAKE AND C O N J U G A T I O N BY A PORCINE, : HEPATOCYTE-BASED BIOARTIFICIAL LIVER (BAL) P.Pazzi*. A.D.Moscioni, J.Rozga, E.Morsiani, A.A.D~metrfou. Dept. of Surgery ' and Liver Support Unit, CedarseSinai Medical Center, Los Angeles, CA, USA. A BAL, consisting of a hollow fiber module containing isolated, microcarrier-attached porcine hepatocytes used as a "bridge!' to liver transplant, has been shown to barry out differentiatedliver functions. Recently (Hepatology 1994; 20:199A), we reported that BAL treatment reduced serum bile acid (BA) levels in patients with fulminant hepatic failure. Aim: To investigate BAL handling of eholic acid. Methods: The BAL, loaded with 2.2 x li38 porcine hepatocytes, separated from the plasma by a semipermeable membrane, was perfused with plasma (fromhealthy donors) Containing either 1 g C i o f [24-14C] Cholate or 1 I.tCi of [24-!4C] taurocholate and supplemented to 100 I.tmol with unlabeled cholate Or taurocholate, respectively. Sample were collected from plasma and from the extrafiber space during a 5-hr perfusion. BAs were separated in accordance with the mode of conjugation (U=unconjugated, G=glyco-conjugated, T=Tauroconjugated, S=Sulphated) on pre-packed silica-based strong'anion exchange cartridges (Bond-Elut SAX)(Chromatographia 1990; 30:377) and radioactivity was determined by liquid scintillation counting. At the end of the perfusion, hepatocytes were collected for radioactivity and protein determinations. Results: Data observed with perfusion with cholateenriched plasma (expressed as % of total radioactivity detected i n each fraction) are reported in the table (n=7): *p<0.001(ANOVA) Fraction Time: 0 1 2 3 4 5 U 97.6+0.2 88.9+0.9 80.3+1.1 78.9_+2.7 76.5_+1.3 76.2+1.4" G 1.2-+0.4 9.6+0.8 18.2-+1,2 20.2-+2.6 22.2_+0.7 22.5+1.3" T 0:6_+0.1 0.9_+0:3 1.3+0.1 2.1-+1.1 1.0+0:4 0.8+1.0 S 0.5-+0.1 0.8+0.3 1.0+0.1 0:7-+0,1 0.3-+0.2 0.4-+0.2 After 3-hr, the G and S fraction of BAs in the extrafiber space were significantly higher than in plasma. During perfusion with taurocholateenriched plasma; a relative decrease in T-fraction and an increase in G; fraction were observed, but the differences were not statistically significant. Cholate was accumulated by hepatocytes to a three-fold louver level than taurocholate, but a significant proportion of radioactivity (>25%) was detected in G-fraction. Conclusions: These results confirm the ability of the BAL to clear BAs from the circulation: porcine hepatocytes accumulate cholate and taurocholate, and are able to conjugate substantial amount of cholic acid. This property could be important clinically in advanced liver disease where bile acid conjugation is impaired.
GASTROENTEROLOGY, Vol. 108, No. 4
• INCREASED NUCLEAR SER1NE PROTEASE ACTIVITY IS A CRITICAL EVENT DURING BILE SALT-INDUCED HEPATOCYTE APOlrrOSIS. T. Patel, S.F. Bronk, G.i Gores, Mayo Clinic, Rochester, MN Hepatoeyte apoptosis induced by bile salts is associated with both enhanced nuclear serine protease and endonuclease activity. However, the sequence of enzymatic events and the molecular mechanisms involved remain obscure. In intact cells, nuclear proteins may need to be disassembled to render DNA susceptible to cleavage by endonucleases and to enable nuclear fragmentation. Thu% our AIMS were to test the ItYFOTItESIS that nuclear serine protease activity precedes and is a prerequisite for DNA deavage and nuclear fragmentation. METHODS: IntraceUular serine protease activity was measured by digitized video microscopy (DVM) using a novel fluorogenic substrate, Cbz-VaI-Leu-Lys-CMAC: Using this probe and DVM, we were able to simultaneously measure nuclear and cytoplasmic protease activity and calculate the nuc!ear/eytoplasmie ratio. DNA strand breaks, a measurement o f endonuclease activity, were quantitated by enzymatically labeling 3'-OH ends of DNA with fluorescein-12-ddUTP. Nuclear fragmentation was quantitated using DAPI and fluorescence microscopy. RESULTS: Apoptosis was induced with 50 jiM glyenehenodeoxycholate (GCDC). ' The nuclear/cytoplasmic serine protease activity ratio reached a maximum 90 rain after addition of GCDC (0.7~0.1 vs. 1.7~-0.2 ratio units, p<0.01). In contrast, nuclear fiagmentation occurred more slowly reaching a maximum 180 rain after addition of GCDC (60.2--1:8.4% vs. <1% in controls, p<0.01). T h e increase in DNA deavage paralleled the increase in nuclear fragmentation during the first 3 hrs of incubation with G-CDC. The serine protease inhibitor, 100 p2CI'i~LCK, reduced nuclear serine protease activity by 67*,4 at 90 minutes. Furthermore, TLCK reduced DNA cleavage by 49i-_4% and nuclear fragmentation by 65*4% after 3 hours of treatment with GCDC. In SUMMARY, that during GCDC-induced apoptosis of hepatocytes l) nuclear serine protease activity increases prior to DNA cleavage and nuclear fragmentation; and 2) inhibition of nuclear serine protease activity prevents DNA cleavage and nuclear fragmentation. CONCLUSION: Increased nuclear serine protease activity appears to be a prerequisite for endonuclease mediated DNA cleavage and for those cellular p r ~ leading to nuclear fragmentation in bile salt-induced hepatoeyte apoptosis.
DETERMINATION OF POTENTIAL NUCLEATING AGENTS IN G A L L B L A D D E R B I L E D U R I N G C H O L E L I T H I A S I S . R. S, P e m s i n g h ~ a n d B.R. M a c P h e r s o n ~. ~School of P o d i a t r i c Medicine, B a r r y University, M i a m i Shores, Florida, 3 3 1 6 1 a n d 2Dept of A n a t o m y a n d Neurobiology, U n i v e r s i t y of Kentucky, Lexington, Ky. 40536. The cholesterol-fed Richardson's ground s q u i r r e l has p r o v e n to be a r e l i a b l e m o d e l for the s t u d y of g a l l s t o n e formation. Male a n d female g r o u n d s q u i r r e l s w e r e d i v i d e d into two groups. The control groups w e r e f e d a con~mercial r a t - c h o w d i e t w h i l e e x p e r i m e n t a l g r o u p s w e r e f e d a 2% c h o l e s t e r o l ~ e n r i c h e d rat c h o w diet. Sample i n t e r v a l s of 6, 12, 18, 24 hours, 3, 5 a n d 7 days were ueed. Gallbladder bile was a s p i r a t e d a n d the pH, c a l c i u m a n d p r o t e i n levels w e r e determined. The b i l e p H f l u c t u a t e d f r o m c o n t r o l s l e v e l s of 7.1 to 6.5 b y 18 hours, 7.6 at d a y 1 a n d 6.4 b y d a y 5. Bile calcium concentration remained w i t h i n n o r m a l range of 19mg/dl i n i t i a l l y b u t b e c a m e s i g n i f i c a n t l y e l e v a t e d to 2 9 m g / d l b y d a y 5. Bile protein concentration exhibited a n i n i t i a l e l e v a t i o n f r o m control v a l u e of 5.5 m g / m l to b e t w e e n 6 - 1 3 m g / m l b y 6 - 1 8 h o u r i n t e r v a l s b u t r e t u r n e d to n o r m a l v a l u e w i t h i n 24 hours. These c h a n g e s o c c u r r e d before and during mucus hypersecretion and cholesterol precipitation, suggesting a p o s s i b l e r o l e as a n u c l e a t i o n cofactor.
Supported by MRC (Alberta, Canada).
(Canada)
and AHFMR
AASLD ABSTRACTS
• THE EFFECT OF A NITRIC OXIDE.SYNTHASE INHIBITOR ON SYSTEMIC HAEMODYNAMICS IN CIRRHOTIC PATIENTS D. Patch. PA McCormick. L Greenslade. R Dick. AK Burroughs. UniversityDept of Medici,e. Royal Free, Hospital. London N'W3. U K Aim: Nitric oxide(NO) has been postulated to mediate the hyperdyfiamic circulation in portal hypertension and cirrhosis. We therefore investigated the effect on systemic haemodynamics of a bolus of a NO synthase inhibitor (LNMMA) in cirrhotic patients. Method: 9 patients (Pughs grade B or C) were studied at the time of routine right heart Swann-Ganz catheterisation prior to liver transplantation. Cardiac output and systemic vascular resistance was calculated by standard flow, dilution. A 10mg bolus of L-NMMA was iniected intravenously in all individuals except patient 5 who received 5mg. Cardiac output studies were perforrded 90-180 secs post-dose. Result s were analysed using a student tr test. Results: (given as mean values) CO SVR PVR mmHg DylLCnLSeC "5 Dyn.cm:sec "5 L/min pre ix)st pro post pro post pre ~os{ 108 112 16 17 72n 75{I 14 57 93 9{) 64o 543 35 18 4 6 92 93 827 7 t 5 53 76 93 97 ' 7 8 750 ~27 34 - 36 83 83 9 9 830 772 29 46 83 93 9 9 761 819 49 47 83 93 10 582 611 62 8 82 80 6 4 1164 1224 43 62 9 100 97 3 4 1333 1315 53 54 No significant difference was detected in the change of any parameters (p>0.1). Discussion: L-NMMA at these doses had little effect on systemm or pulmonary haemodynamics in cirrhotic patients No adverse effects were noted. These results suggest that nitric oxide may not be the principle mediator of the vasodilated state of end-stage cirrhosis Further studies with higher doses of L-NMMA are required BP
CHOLATE UPTAKE AND C O N J U G A T I O N BY A PORCINE, : HEPATOCYTE-BASED BIOARTIFICIAL LIVER (BAL) P.Pazzi*. A.D.Moscioni, J.Rozga, E.Morsiani, A.A.D~metrfou. Dept. of Surgery ' and Liver Support Unit, CedarseSinai Medical Center, Los Angeles, CA, USA. A BAL, consisting of a hollow fiber module containing isolated, microcarrier-attached porcine hepatocytes used as a "bridge!' to liver transplant, has been shown to barry out differentiatedliver functions. Recently (Hepatology 1994; 20:199A), we reported that BAL treatment reduced serum bile acid (BA) levels in patients with fulminant hepatic failure. Aim: To investigate BAL handling of eholic acid. Methods: The BAL, loaded with 2.2 x li38 porcine hepatocytes, separated from the plasma by a semipermeable membrane, was perfused with plasma (fromhealthy donors) Containing either 1 g C i o f [24-14C] Cholate or 1 I.tCi of [24-!4C] taurocholate and supplemented to 100 I.tmol with unlabeled cholate Or taurocholate, respectively. Sample were collected from plasma and from the extrafiber space during a 5-hr perfusion. BAs were separated in accordance with the mode of conjugation (U=unconjugated, G=glyco-conjugated, T=Tauroconjugated, S=Sulphated) on pre-packed silica-based strong'anion exchange cartridges (Bond-Elut SAX)(Chromatographia 1990; 30:377) and radioactivity was determined by liquid scintillation counting. At the end of the perfusion, hepatocytes were collected for radioactivity and protein determinations. Results: Data observed with perfusion with cholateenriched plasma (expressed as % of total radioactivity detected i n each fraction) are reported in the table (n=7): *p<0.001(ANOVA) Fraction Time: 0 1 2 3 4 5 U 97.6+0.2 88.9+0.9 80.3+1.1 78.9_+2.7 76.5_+1.3 76.2+1.4" G 1.2-+0.4 9.6+0.8 18.2-+1,2 20.2-+2.6 22.2_+0.7 22.5+1.3" T 0:6_+0.1 0.9_+0:3 1.3+0.1 2.1-+1.1 1.0+0:4 0.8+1.0 S 0.5-+0.1 0.8+0.3 1.0+0.1 0:7-+0,1 0.3-+0.2 0.4-+0.2 After 3-hr, the G and S fraction of BAs in the extrafiber space were significantly higher than in plasma. During perfusion with taurocholateenriched plasma; a relative decrease in T-fraction and an increase in G; fraction were observed, but the differences were not statistically significant. Cholate was accumulated by hepatocytes to a three-fold louver level than taurocholate, but a significant proportion of radioactivity (>25%) was detected in G-fraction. Conclusions: These results confirm the ability of the BAL to clear BAs from the circulation: porcine hepatocytes accumulate cholate and taurocholate, and are able to conjugate substantial amount of cholic acid. This property could be important clinically in advanced liver disease where bile acid conjugation is impaired.
GASTROENTEROLOGY, Vol. 108, No. 4
• INCREASED NUCLEAR SER1NE PROTEASE ACTIVITY IS A CRITICAL EVENT DURING BILE SALT-INDUCED HEPATOCYTE APOlrrOSIS. T. Patel, S.F. Bronk, G.i Gores, Mayo Clinic, Rochester, MN Hepatoeyte apoptosis induced by bile salts is associated with both enhanced nuclear serine protease and endonuclease activity. However, the sequence of enzymatic events and the molecular mechanisms involved remain obscure. In intact cells, nuclear proteins may need to be disassembled to render DNA susceptible to cleavage by endonucleases and to enable nuclear fragmentation. Thu% our AIMS were to test the ItYFOTItESIS that nuclear serine protease activity precedes and is a prerequisite for DNA deavage and nuclear fragmentation. METHODS: IntraceUular serine protease activity was measured by digitized video microscopy (DVM) using a novel fluorogenic substrate, Cbz-VaI-Leu-Lys-CMAC: Using this probe and DVM, we were able to simultaneously measure nuclear and cytoplasmic protease activity and calculate the nuc!ear/eytoplasmie ratio. DNA strand breaks, a measurement o f endonuclease activity, were quantitated by enzymatically labeling 3'-OH ends of DNA with fluorescein-12-ddUTP. Nuclear fragmentation was quantitated using DAPI and fluorescence microscopy. RESULTS: Apoptosis was induced with 50 jiM glyenehenodeoxycholate (GCDC). ' The nuclear/cytoplasmic serine protease activity ratio reached a maximum 90 rain after addition of GCDC (0.7~0.1 vs. 1.7~-0.2 ratio units, p<0.01). In contrast, nuclear fiagmentation occurred more slowly reaching a maximum 180 rain after addition of GCDC (60.2--1:8.4% vs. <1% in controls, p<0.01). T h e increase in DNA deavage paralleled the increase in nuclear fragmentation during the first 3 hrs of incubation with G-CDC. The serine protease inhibitor, 100 p2CI'i~LCK, reduced nuclear serine protease activity by 67*,4 at 90 minutes. Furthermore, TLCK reduced DNA cleavage by 49i-_4% and nuclear fragmentation by 65*4% after 3 hours of treatment with GCDC. In SUMMARY, that during GCDC-induced apoptosis of hepatocytes l) nuclear serine protease activity increases prior to DNA cleavage and nuclear fragmentation; and 2) inhibition of nuclear serine protease activity prevents DNA cleavage and nuclear fragmentation. CONCLUSION: Increased nuclear serine protease activity appears to be a prerequisite for endonuclease mediated DNA cleavage and for those cellular p r ~ leading to nuclear fragmentation in bile salt-induced hepatoeyte apoptosis.
DETERMINATION OF POTENTIAL NUCLEATING AGENTS IN G A L L B L A D D E R B I L E D U R I N G C H O L E L I T H I A S I S . R. S, P e m s i n g h ~ a n d B.R. M a c P h e r s o n ~. ~School of P o d i a t r i c Medicine, B a r r y University, M i a m i Shores, Florida, 3 3 1 6 1 a n d 2Dept of A n a t o m y a n d Neurobiology, U n i v e r s i t y of Kentucky, Lexington, Ky. 40536. The cholesterol-fed Richardson's ground s q u i r r e l has p r o v e n to be a r e l i a b l e m o d e l for the s t u d y of g a l l s t o n e formation. Male a n d female g r o u n d s q u i r r e l s w e r e d i v i d e d into two groups. The control groups w e r e f e d a con~mercial r a t - c h o w d i e t w h i l e e x p e r i m e n t a l g r o u p s w e r e f e d a 2% c h o l e s t e r o l ~ e n r i c h e d rat c h o w diet. Sample i n t e r v a l s of 6, 12, 18, 24 hours, 3, 5 a n d 7 days were ueed. Gallbladder bile was a s p i r a t e d a n d the pH, c a l c i u m a n d p r o t e i n levels w e r e determined. The b i l e p H f l u c t u a t e d f r o m c o n t r o l s l e v e l s of 7.1 to 6.5 b y 18 hours, 7.6 at d a y 1 a n d 6.4 b y d a y 5. Bile calcium concentration remained w i t h i n n o r m a l range of 19mg/dl i n i t i a l l y b u t b e c a m e s i g n i f i c a n t l y e l e v a t e d to 2 9 m g / d l b y d a y 5. Bile protein concentration exhibited a n i n i t i a l e l e v a t i o n f r o m control v a l u e of 5.5 m g / m l to b e t w e e n 6 - 1 3 m g / m l b y 6 - 1 8 h o u r i n t e r v a l s b u t r e t u r n e d to n o r m a l v a l u e w i t h i n 24 hours. These c h a n g e s o c c u r r e d before and during mucus hypersecretion and cholesterol precipitation, suggesting a p o s s i b l e r o l e as a n u c l e a t i o n cofactor.
Supported by MRC (Alberta, Canada).
(Canada)
and AHFMR