- Email: [email protected]
Highly effective enteral treatment of unconjugated hyperbilirubinemia in Gunn rats
189
Conclusions: The absence of PC-TP is associated with reduced hepatic cholesterol catabolism to bile salts without changes in cholesterol synthesis. As indicated by preserved UOPL ratios, compensatory upregulation of Acat prevents membrane accumulation of UC. In response to HC feeding, further reductions in bile salt synthesis are not compensated m KO mice either by upregulation of Acat or by sufficiem increases in hepatic PL to prevent accumulation of UC in membranes, as evidenced by increased UC/PL ratios. We speculate that regulation of cholesterol homeostasis is a critical function of PC-TP in liver, particularly in the setting of a dietary cholesterol challenge.
Highly Effective Enteral Treatment Of Uneonjugated Hyperbilirubinemia In Gunn Rats Anja M. Hatkamp, Rick Havinga, Maarten Sinaasappel, Ronald P. J. Oude Elferink, Henkjan J. Verkade BACKGROUND: We aim to develop a treatment of unconjugated hyperbifirubinemia that is based on enteral administration, and has a higher efficacy than phototherapy (PT). In the Gunn rat model of unconjugated hyperbilirubinemia, dietary supplementation with calcium phosphate (CAP) or with the lipasa inhibitor orlistat (Orl) decreases plasma unconjugated bilirubin (UCB) concentrations: In Gunn rats, Orl is equally effective as PT (Pediatr Res 2002;51:1912A). It is unknown whether the effects of Orl and CaP are influenced by dietary fat content, and whether their combined treatment (Orl + CaP) is superior to PT. OBJECTIVE: To determine in Gunn rats the effects of Orl, CaP, and Off + CaP on plasma UCB concentrations during a low-fat (LF) and a high-fat (HF) diet. METHODS: Adult male Gunn rats (45 per group) were fed a LF diet (15 energy% fat) for 3 weeks, followed by a HF diet (35 energy% fat) for 3 weeks. Diets were either not supplemented (controls), or supplemented with Orl (200 mg/kg chow), CaP (20 g/kg chow), or Orl+ CaP. Separate Gunn rats on the HF diet received continuous PT (380-480 nm, 9 gW/cm2/nm) for 2 weeks. Plasma UCB concentrations and fecal fat excretion (72h) were determined by HPLC and GC, respectively. RESULTS: In all Gunn rats, except CaP-supplemented, plasma UCB concentrations decreased profoundly by the change from LF to HF diet (p<0.01). In controls, plasma UCB decreased from 248+- 31 ~tM to 135 +- 10 ~M (LF/HF, resp.; mean+_SD; p<0.001), and appeared inversely correlated with fecal fat excretion (LF, 0.07+_0.03; HF, 0.74_+0.12 mmol/24h; r-0.96, p<0.001). During either diet, supplementation with Orl or CaP decreased plasma UCB compared vclth controls (LF, Or1-30%, p
wr. Chow KO" Chow
WI"" HC KO" HC
~IAt
81G
10.1,5.8
20,2
2,2,0.2
919
5,6 0.7"
165!64*
1.9!0.2
23' 3T
5.8!0.91"
91 !141"
17.3!1.01"
5,90,41"
26,4!t.1
0.38 *0.041'
24:31"
3,6'0.5*1"
11856
22,1!t.71"
12,3!1.4"T
41.5"1.4'1"
0.57!0,06'7
~
TC
UC
PL
UC/PL
1.0,0.2
24,0'.1.7
0.18'0r01
1.3!0,1"
16.7+1.0"
0.18!0,01
*p
192
A New Theory to Explain Variceal Bleeding in Patients with Portal Hypertension: Peristaltic Contractions Generate Supernormal Pressures in the Distal Portion of Esophageal Varices Larry S. Miller, Vlnod K Thangada, Joseph K. Kim, Qing Dai, Elan S. Miller, Jasneet Gandegok, Gustavo Torres, Beje Thomas, Justin J. Harberson, Carson Schneck, Martin Black Purpose: To measure intravariceal pressure during peristaltic contraction. Method: 9 patients with esophageal varices underwent simultaneous esophageal ultrasound (US) and manometry. Each patient was given 5cc boluses of water and asked to swallow. The esophageal luminal pressure at which the varix closed and opened on US was measured. The esophageal pressure at variceal closure equals the intravariceal pressure. Results: The closing pressure (mean = 43.82 _+ 16.77 mmHg) was sigmficantly higher than the opening pressures (mean = 11.56 _+ 25.56mmHg, p = 0.0001). Closing pressures >80mmHg were commonly generated in varices during peristaltic contractions (11%) and closing pressures > 100mmHg were not unusual (4.5%). There was a strong correlation between the peak peristaltic pressures and the closing pressures (r between 0.78 and 0.99). On clinical fonow-up 4 patients (44%) bled an average of 11.5 + 7.55 months after the US/manometry test. The patients that bled had a significantly higher mean peak closing pressure (83.15 +_ 18.38 mmHg) than the patients that did not bleed (53.2 + 7.67 mmHg.) after a mean of 16.4 _+ 0.89 months, (p < 0.04). Using 61mmHg as a cutoff for mean peak closing pressure, the sensitivity, and specificity, positive and negative predictive value for predicting future variceal bleeding were each 100%. Conclusion: Blood in esophageal varices is pushed in a proximal to distal direction during peristakic contraction essentially reversing the normal flow of blood. During the peristaltic contraction the blood encounters resistance vessels at the gastroesophageal junction (palisade and bar type vessels). This generates variceal pressures, in the distal esophagus, that are far in excess of the static pressures previously measured in esophageal varices (Ohm's law; Ap = R x Q). This accounts for the high closing pressures. The varices open at a significantly lower pressure than the closing pressure when the blood refills the varices from the stomach. The higher the peak amplitude of the peristaltic comraction (peak pressure) the higher the closing pressure of the varix The closing pressures in patients that experience future variceal bleeding are significantly higher than the closing pressures in patients that do not (p< 0.04). Patients with a mean peak closing pressure > 61 mmHg are more likely to bleed (p<0.003). The accuracy of predicting future variceal bleeding in this study was 100%.
190
FXR Is the Dominant Regulator of CYP7A1 in the Rat Guorong Xu, Luxing Pan, Quan Shang, Hal Li, Stephen Tim, Gerald Salen Aim: CYP7A1, the rate-limiting enzyme for classic bile acid synthesis is up-regulated in rats but down-regulated in rabbits after cholesterol feedmg. To clarify the mechanism responsible for this different regulation, the effects of dietary cholesterol (Ch) and cholic acid (CA) on FXR and LXR activation which regulate CYP7A1 were investigated in rats. Method: Four groups ( n = 6 per group) of Sprague Dawley rats were studied: controls, fed 2% Ch, fed 2% Ch + 1% CA, and fed 1% CA. Changes in the mRNA expression of short heterodimer partner (SHP) and bile salt export pump (BSEP), two target genes of FXR were determined to indicate FXR activation state and the expression of ABCA1, the target gene of LXR, reflected LXR activation. Results: After 1 week of treatment, CYP7A1 mRNA increased 95% (p=0.01) in rats fed Ch but decreased 60% (p<0.05) in rats fed CA. Combining Ch with CA did not increase CYP7A1 mRNA above baseline. In Ch-fed rats, SHP mRNA did not change but increased 82% (p<0.05%) and 2.6-fold (p
193
HDL Cholesterol is an Indicator of Liver Function and Prognosis in Advanced Cirrhosis Adil Habib, Souheil G. Abou-Assi, Anastasios A. Mihas, William M. Pandak, Edith Gavis, Douglas M. Heuman Background: The liver plays a cemral role in the production and degradation of lipoproteins (LPs). Decreases in circulating cholesterol and in apo-LPs, especially A1, have been noted in advanced liver disease. We hypothesized that routine analysis of circulating LP cholesterol fractions may be useful in assessing severity and prognosis of cirrhosis. Methods: Subjects included 930 veterans with established cirrhosis who were followed in our liver clinics or referred for consideration of liver transplantation between 1997 and 2002. Of these, LP cholesterol data were available for 265. Population was 98% male, and causes of cirrhosis were alcohol and/or hepatitis C in 88%. Plasma cholesterol (Ch) was determined using cholesterol oxidase; HDL and non-HDL fractions were determined following precipitation with MgC12/dextran, and VLDL fraction was estimated as 02 x triglyceride. Concurrent albumin, bilimhin, INR, creatinine, and MELD score were available in 204 patients. Outcome (survival from date of most recent complete laboratory results, censored at transplantation) was confirmed through VA electronic records and through the social security death index Results: Both HDL and total Ch correlated inversely with serum albumin (p < 0.001) and also with INR (p < 0.05). LDL Ch did not correlate with any liver function test. VLDL Ch correlated significantly only with INR. Mortality at 90, 180 and 365 days was 18/233, 21/ 200 and 40/135, respectively. By univariate analysis, death at all three points was associated with significantly lower initial levels of HDL, VLDL and total Ch, but not LDL Ch. Of the lipoproteins, HDL was clearly the best predictor of mortality in cirrhosis; concordance statistics for HDL Ch as a predictor of mortality at 90. 180 and 360 days were (mean + SE) .82+_.06, . 8 6 +- .05, and .76+_ .05, respectively, compared to 66_+ .08, .51 +_ .08 and .50 + .06 for non-HDL Ch By multivariate logistic regression (including all Lp fractions and liver function tests), independent predictors of survival at 90 d and 180 d were MELD score and albumin. In contrast for 1 year su~val, HDL Ch was the only independent predictor
191
Phosphatidylcholine Transfer Protein (PC-TP) Regulates Hepatic Cholesterol Metabolism in Mice Michele K. Wu, Suresh K. Yadav, David E. Cohen
Background: PC-TP is a member of the steroidoganlc acute regulatory transfer (START) domain superfamily of proteins and is highly expressed iu hver. Although its function is not known with certainty, we have shown that PC-TP may facilitate export of cholesterol from liver into plasma by promoting high density fipoprotein (HDL) formation. Aim: To further explore a role for PC-TP in h~patic cholesterol metabolism. Methods: Eight-week old male Pctp(-/-) (KO) and wild type (WT) mice were fed a chow (4% fat, <0.02% cholesterol; n >5/group) or a high cholesterol (HC; 15% fat, 1.25% cholesterol, 0.5% cholate; n > 5/ group) diet. After 7 d, we determined hepatic activities of HMG-CoA reductase (HMR), cholesterol 7a-hydroxylase (Cyp7A1) and acyl CoA:cholesterol acyltransferase (Acat), as well as hepatic concentrations of phospholipids (PL) and total (TC) and unesterified (UC) cholesterol. Results: Compared with chow-fed WT mice, HMR activity was unchanged, Cyp7A1 activity was lower and Acat activity was higher in KO mice. Whereas TC was similar, UC was reduced in livers of KO mice. PL was also reduced, so that the UC/PL ratio remained unchanged. HC feeding downregulated HMR and Cyp7A1 in both WT and KO mice, whereas Acat was increased in WT but not KO mice. HC feeding increased UC to a greater extent in KO mice, and despite higher PL, the UC/PL ratio was also increased.
AGA Abstracts
HEiR
A-28
Conclusions: The absence of PC-TP is associated with reduced hepatic cholesterol catabolism to bile salts without changes in cholesterol synthesis. As indicated by preserved UOPL ratios, compensatory upregulation of Acat prevents membrane accumulation of UC. In response to HC feeding, further reductions in bile salt synthesis are not compensated m KO mice either by upregulation of Acat or by sufficiem increases in hepatic PL to prevent accumulation of UC in membranes, as evidenced by increased UC/PL ratios. We speculate that regulation of cholesterol homeostasis is a critical function of PC-TP in liver, particularly in the setting of a dietary cholesterol challenge.
Highly Effective Enteral Treatment Of Uneonjugated Hyperbilirubinemia In Gunn Rats Anja M. Hatkamp, Rick Havinga, Maarten Sinaasappel, Ronald P. J. Oude Elferink, Henkjan J. Verkade BACKGROUND: We aim to develop a treatment of unconjugated hyperbifirubinemia that is based on enteral administration, and has a higher efficacy than phototherapy (PT). In the Gunn rat model of unconjugated hyperbilirubinemia, dietary supplementation with calcium phosphate (CAP) or with the lipasa inhibitor orlistat (Orl) decreases plasma unconjugated bilirubin (UCB) concentrations: In Gunn rats, Orl is equally effective as PT (Pediatr Res 2002;51:1912A). It is unknown whether the effects of Orl and CaP are influenced by dietary fat content, and whether their combined treatment (Orl + CaP) is superior to PT. OBJECTIVE: To determine in Gunn rats the effects of Orl, CaP, and Off + CaP on plasma UCB concentrations during a low-fat (LF) and a high-fat (HF) diet. METHODS: Adult male Gunn rats (45 per group) were fed a LF diet (15 energy% fat) for 3 weeks, followed by a HF diet (35 energy% fat) for 3 weeks. Diets were either not supplemented (controls), or supplemented with Orl (200 mg/kg chow), CaP (20 g/kg chow), or Orl+ CaP. Separate Gunn rats on the HF diet received continuous PT (380-480 nm, 9 gW/cm2/nm) for 2 weeks. Plasma UCB concentrations and fecal fat excretion (72h) were determined by HPLC and GC, respectively. RESULTS: In all Gunn rats, except CaP-supplemented, plasma UCB concentrations decreased profoundly by the change from LF to HF diet (p<0.01). In controls, plasma UCB decreased from 248+- 31 ~tM to 135 +- 10 ~M (LF/HF, resp.; mean+_SD; p<0.001), and appeared inversely correlated with fecal fat excretion (LF, 0.07+_0.03; HF, 0.74_+0.12 mmol/24h; r-0.96, p<0.001). During either diet, supplementation with Orl or CaP decreased plasma UCB compared vclth controls (LF, Or1-30%, p
wr. Chow KO" Chow
WI"" HC KO" HC
~IAt
81G
10.1,5.8
20,2
2,2,0.2
919
5,6 0.7"
165!64*
1.9!0.2
23' 3T
5.8!0.91"
91 !141"
17.3!1.01"
5,90,41"
26,4!t.1
0.38 *0.041'
24:31"
3,6'0.5*1"
11856
22,1!t.71"
12,3!1.4"T
41.5"1.4'1"
0.57!0,06'7
~
TC
UC
PL
UC/PL
1.0,0.2
24,0'.1.7
0.18'0r01
1.3!0,1"
16.7+1.0"
0.18!0,01
*p
192
A New Theory to Explain Variceal Bleeding in Patients with Portal Hypertension: Peristaltic Contractions Generate Supernormal Pressures in the Distal Portion of Esophageal Varices Larry S. Miller, Vlnod K Thangada, Joseph K. Kim, Qing Dai, Elan S. Miller, Jasneet Gandegok, Gustavo Torres, Beje Thomas, Justin J. Harberson, Carson Schneck, Martin Black Purpose: To measure intravariceal pressure during peristaltic contraction. Method: 9 patients with esophageal varices underwent simultaneous esophageal ultrasound (US) and manometry. Each patient was given 5cc boluses of water and asked to swallow. The esophageal luminal pressure at which the varix closed and opened on US was measured. The esophageal pressure at variceal closure equals the intravariceal pressure. Results: The closing pressure (mean = 43.82 _+ 16.77 mmHg) was sigmficantly higher than the opening pressures (mean = 11.56 _+ 25.56mmHg, p = 0.0001). Closing pressures >80mmHg were commonly generated in varices during peristaltic contractions (11%) and closing pressures > 100mmHg were not unusual (4.5%). There was a strong correlation between the peak peristaltic pressures and the closing pressures (r between 0.78 and 0.99). On clinical fonow-up 4 patients (44%) bled an average of 11.5 + 7.55 months after the US/manometry test. The patients that bled had a significantly higher mean peak closing pressure (83.15 +_ 18.38 mmHg) than the patients that did not bleed (53.2 + 7.67 mmHg.) after a mean of 16.4 _+ 0.89 months, (p < 0.04). Using 61mmHg as a cutoff for mean peak closing pressure, the sensitivity, and specificity, positive and negative predictive value for predicting future variceal bleeding were each 100%. Conclusion: Blood in esophageal varices is pushed in a proximal to distal direction during peristakic contraction essentially reversing the normal flow of blood. During the peristaltic contraction the blood encounters resistance vessels at the gastroesophageal junction (palisade and bar type vessels). This generates variceal pressures, in the distal esophagus, that are far in excess of the static pressures previously measured in esophageal varices (Ohm's law; Ap = R x Q). This accounts for the high closing pressures. The varices open at a significantly lower pressure than the closing pressure when the blood refills the varices from the stomach. The higher the peak amplitude of the peristaltic comraction (peak pressure) the higher the closing pressure of the varix The closing pressures in patients that experience future variceal bleeding are significantly higher than the closing pressures in patients that do not (p< 0.04). Patients with a mean peak closing pressure > 61 mmHg are more likely to bleed (p<0.003). The accuracy of predicting future variceal bleeding in this study was 100%.
190
FXR Is the Dominant Regulator of CYP7A1 in the Rat Guorong Xu, Luxing Pan, Quan Shang, Hal Li, Stephen Tim, Gerald Salen Aim: CYP7A1, the rate-limiting enzyme for classic bile acid synthesis is up-regulated in rats but down-regulated in rabbits after cholesterol feedmg. To clarify the mechanism responsible for this different regulation, the effects of dietary cholesterol (Ch) and cholic acid (CA) on FXR and LXR activation which regulate CYP7A1 were investigated in rats. Method: Four groups ( n = 6 per group) of Sprague Dawley rats were studied: controls, fed 2% Ch, fed 2% Ch + 1% CA, and fed 1% CA. Changes in the mRNA expression of short heterodimer partner (SHP) and bile salt export pump (BSEP), two target genes of FXR were determined to indicate FXR activation state and the expression of ABCA1, the target gene of LXR, reflected LXR activation. Results: After 1 week of treatment, CYP7A1 mRNA increased 95% (p=0.01) in rats fed Ch but decreased 60% (p<0.05) in rats fed CA. Combining Ch with CA did not increase CYP7A1 mRNA above baseline. In Ch-fed rats, SHP mRNA did not change but increased 82% (p<0.05%) and 2.6-fold (p
193
HDL Cholesterol is an Indicator of Liver Function and Prognosis in Advanced Cirrhosis Adil Habib, Souheil G. Abou-Assi, Anastasios A. Mihas, William M. Pandak, Edith Gavis, Douglas M. Heuman Background: The liver plays a cemral role in the production and degradation of lipoproteins (LPs). Decreases in circulating cholesterol and in apo-LPs, especially A1, have been noted in advanced liver disease. We hypothesized that routine analysis of circulating LP cholesterol fractions may be useful in assessing severity and prognosis of cirrhosis. Methods: Subjects included 930 veterans with established cirrhosis who were followed in our liver clinics or referred for consideration of liver transplantation between 1997 and 2002. Of these, LP cholesterol data were available for 265. Population was 98% male, and causes of cirrhosis were alcohol and/or hepatitis C in 88%. Plasma cholesterol (Ch) was determined using cholesterol oxidase; HDL and non-HDL fractions were determined following precipitation with MgC12/dextran, and VLDL fraction was estimated as 02 x triglyceride. Concurrent albumin, bilimhin, INR, creatinine, and MELD score were available in 204 patients. Outcome (survival from date of most recent complete laboratory results, censored at transplantation) was confirmed through VA electronic records and through the social security death index Results: Both HDL and total Ch correlated inversely with serum albumin (p < 0.001) and also with INR (p < 0.05). LDL Ch did not correlate with any liver function test. VLDL Ch correlated significantly only with INR. Mortality at 90, 180 and 365 days was 18/233, 21/ 200 and 40/135, respectively. By univariate analysis, death at all three points was associated with significantly lower initial levels of HDL, VLDL and total Ch, but not LDL Ch. Of the lipoproteins, HDL was clearly the best predictor of mortality in cirrhosis; concordance statistics for HDL Ch as a predictor of mortality at 90. 180 and 360 days were (mean + SE) .82+_.06, . 8 6 +- .05, and .76+_ .05, respectively, compared to 66_+ .08, .51 +_ .08 and .50 + .06 for non-HDL Ch By multivariate logistic regression (including all Lp fractions and liver function tests), independent predictors of survival at 90 d and 180 d were MELD score and albumin. In contrast for 1 year su~val, HDL Ch was the only independent predictor
191
Phosphatidylcholine Transfer Protein (PC-TP) Regulates Hepatic Cholesterol Metabolism in Mice Michele K. Wu, Suresh K. Yadav, David E. Cohen
Background: PC-TP is a member of the steroidoganlc acute regulatory transfer (START) domain superfamily of proteins and is highly expressed iu hver. Although its function is not known with certainty, we have shown that PC-TP may facilitate export of cholesterol from liver into plasma by promoting high density fipoprotein (HDL) formation. Aim: To further explore a role for PC-TP in h~patic cholesterol metabolism. Methods: Eight-week old male Pctp(-/-) (KO) and wild type (WT) mice were fed a chow (4% fat, <0.02% cholesterol; n >5/group) or a high cholesterol (HC; 15% fat, 1.25% cholesterol, 0.5% cholate; n > 5/ group) diet. After 7 d, we determined hepatic activities of HMG-CoA reductase (HMR), cholesterol 7a-hydroxylase (Cyp7A1) and acyl CoA:cholesterol acyltransferase (Acat), as well as hepatic concentrations of phospholipids (PL) and total (TC) and unesterified (UC) cholesterol. Results: Compared with chow-fed WT mice, HMR activity was unchanged, Cyp7A1 activity was lower and Acat activity was higher in KO mice. Whereas TC was similar, UC was reduced in livers of KO mice. PL was also reduced, so that the UC/PL ratio remained unchanged. HC feeding downregulated HMR and Cyp7A1 in both WT and KO mice, whereas Acat was increased in WT but not KO mice. HC feeding increased UC to a greater extent in KO mice, and despite higher PL, the UC/PL ratio was also increased.
AGA Abstracts
HEiR
A-28