- Email: [email protected]
[209] EX VIVO ENDOTOXIN REMOVAL FROM PLASMA RESTORES NEUTROPHIL FUNCTION
02A. CIRRHOSIS AND COMPLICATIONS A) PATHOPHYSIOLOGY -
Aim: To examine differences in clearance and metabolism of intravenously injected 13N-ammonia between cirrhosis patients with clinical hepatic encephalopathy (HE), cirrhosis patients without HE, and healthy controls. Methods: Twelve cirrhosis patients with HE, three without HE, and six healthy controls were included. 500 MBq of 13N-ammonia was injected i.v. and a series of arterial blood samples was taken over 30 minutes to measure total concentration of 13N-radioactivity and fractions of 13NNH3, 13N-glutamine, 13N-urea, and 13N-acidic amino acids (negligible). Arterial pH and ammonia were measured and liver function estimated by GEC. Results: We found no correlation between GEC and the degree of HE, nor between GEC and blood ammonia concentration. Cirrhosis patients without HE had the highest initial clearance of I3N-ammonia and cirrhosis patients with HE the lowest initial clearance. AUCs of 13N-ammonia was significantly higher in patients with HE compared to the other two groups, showing a decreased ammonia blood clearance. Total I3N-radioactivity concentration in healthy controls and patients without HE ended at similar levels, but at a higher level in patients with HE. Arterial pH in HE patients was elevated. Lower pH in patients without HE could make clearance of ammonia more dependent on regulated membrane transport proteins and partly explain why some cirrhosis patients develop symptoms of HE. High turnover of ammonia to glutamine in muscles might reduce the risk of developing HE and increased deamination of glutamine might increase the risk of HE. This is supported by the finding of lower late clearance of I3N-ammonia in patients with HE compared to patients without HE and healthy controls but no significant difference in the early clearance. Conclusions: Patients with liver cirrhosis and HE clear blood ammonia much slower than patients with cirrhosis but no HE and healthy controls. Our data support other findings that pH and muscular glutamine production seem to influence development of HE.
12091 EX VlVO ENDOTOXIN REMOVAL FROM PLASMA RESTORES NEUTROPHIL FUNCTION
V Stadlbauer, R.P. Mookerjee, S. Lidder, S.J. Hodges, N.A. Davies, R. Williams, R. Jalan. The 7JCL Institute ~ f ’ H e p t o l o g yLondon, , UK E-mail: [email protected] Background: In patients with alcoholic hepatitis, organ failure and mortality are often related to infection suggesting a state of ‘immune failure’. We previously demonstrated that neutrophils from patients with alcoholic hepatitis are fully activated (high resting burst) and show decreased phagocytic capacity. High resting burst and decreased phagocytosis was associated with infection, organ failure and mortality. Therefore we aimed to investigate whether endotoxin can cause these changes in neutrophils and whether endotoxin removal from patients’ plasma can restore neutrophil function ex vivo. Method: Peripheral blood neutrophils were isolated from patients or healthy volunteers and incubated with endotoxin-spiked plasma from controls or plasma from patients. Neutrophil oxidative burst activity and phagocytic capacity was assayed by FACS analysis using the Phagoburst test and Phagotest (Orpegen). Endotoxin was removed either by passing the plasma over a polymixin B containing column (Detoxi-Gel, Pierce Biotechnology) or incubation with an LPS-neutralizing anti-CD14 antibody (Clone llD18) Results: Incubating neutrophil from healthy volunteers with increasing endotoxin concentrations (50-200ng/ml) resulted in a concentrationdependent increase in resting burst (n = 5 , p < 0.0001). Incubating patients’ neutrophils with endotoxin reduced phagocytosis by 20% (n = 8, p 1 0.05). When plasma was passed over Detoxi-Gel columns, resting burst was reduced by 32% (p 0.00 I , n = 9) and phagocytosis increased by 3 1 % (p 1 0.05, n = 1 I ) . When plasma from patients with low resting burst (n = 8) and normal plasma ( n = 5 ) was passed over the column, no changes in resting burst or phagocytosis were observed. Incubation with an anti-CD 14 antibody prevented the induction of increased burst in normal neutrophils by high burst patient plasma (p < 0.001, n = 7 ) and increased phagocytosis
S87
by 20% (p 0.05, n = 1 I ) , whilst having no effect on neutrophils incubated with low burst (n = 8) or healthy control plasma (n = 3). Discussion: Our data support the notion that endotoxin may cause functional changes in neutrophils similar to those observed in patients with alcoholic hepatitis and that ex vivo removal of endotoxin restores neutrophil function. This novel observation may have important therapeutic implications: Patients with high resting burst and decreased phagocytic capacity, who are at increased risk for infection, might benefit from endotoxin removal strategies rather than from treatment with steroids.
12101 EVIDENCE FOR A PATHOGENETIC ROLE OF ASYMMETRIC DIMETHYLARGININE (ADMA) IN CIRRHOTIC PORTAL HYPERTENS1ON F. Vizzutti’, R.G. Romanelli’, U. Arena’, L. Rega2, M. Brogi’, F. Marra’, M. Foschi’, R. Tarquini’, G. Laffi’, M. Pinzani’. ‘Dipurtimrnto di Mrdicinu Internu Uniuersitu di Firrnzr, Firenzr; 2Dipurtimmto di Rudiologiu, Aziendu Osprduliero Uniuer~xituriuCureggi Firrnzr, Firrnze, Italy E-mail: [email protected]
Background: Chronic liver diseases (CLD) are frequently complicated by portal hypertension, an important component of which is the increased intrahepatic vascular resistance, in part related to intra-hepatic endothelial dysfunction. ADMA, an endogenous inhibitor of NO synthase (NOS), has emerged in a variety of cardiovascular disease as a mediator of endothelial dysfunction at relatively low plasma concentrations. Aim: Although elevated ADMA plasma level have been reported in endstage CLD, there is no clear evidence of ADMA involvement in the pathogenesis of portal hypertension in humans. We therefore investigated the possible implication of ADMA in this clinical setting. Patients and Methods: We studied 39 consecutive patients with HCV related CLD at different stage of fibrotic evolution: 34 cirrhotic, and 5 non-cirrhotic patients (METAVIR F2 to F3). All patients underwent HVPG measurements, and simultaneous blood samples were taken from the hepatic vein and pulmonary artery, for ADMA and NOx plasma levels determinations. Exclusion criteria were: cardiopulmonary disease, arterial hypertension, diabetes mellitus, hypercholesterolemia, hyperomocysteinemia, acute and chronic renal failure, chronic inflammatory diseases, recent active alcohol intake, use of vasoactive drugs andor NSAIDs. Results: A positive correlation among HVPG and ADMA concentrations in hepatic veins (ADMA-h) and pulmonary artery (ADMA-p) (r=0.77, p iO.OOOO1 and r=0.56, p < 0.00001, respectively) was found. Moreover, a negative correlation between HVPG and NOx concentrations in hepatic vein (NO-h) (r=-0.50, p <0.005), and between ADMA-h and NO-h was observed (r=-0.40, p < 0.02). ADMA-p levels (0.55f0.13 LimoliL) were significantly higher than ADMA-h ones (0.47&0.09 pmol/L) (p < 0.00001). Systemic and hepatic vein ADMA and NOx concentrations did not correlate either with Child-Pugh class or score in cirrhotic patients. However, when patients were classified according to Child-Pugh score 1 7 and 28, a correlation with ADMA-p levels was 0.02). observed (p i Conclusions: ADMA-h is directly related to portal pressure and inversely related to NO-h, thus indicating that ADMA may play a pathophysiological role contributing to the relative intra-hepatic NO deficiency typical of the so-called cirrhotic endothelial dysfunction.
Aim: To examine differences in clearance and metabolism of intravenously injected 13N-ammonia between cirrhosis patients with clinical hepatic encephalopathy (HE), cirrhosis patients without HE, and healthy controls. Methods: Twelve cirrhosis patients with HE, three without HE, and six healthy controls were included. 500 MBq of 13N-ammonia was injected i.v. and a series of arterial blood samples was taken over 30 minutes to measure total concentration of 13N-radioactivity and fractions of 13NNH3, 13N-glutamine, 13N-urea, and 13N-acidic amino acids (negligible). Arterial pH and ammonia were measured and liver function estimated by GEC. Results: We found no correlation between GEC and the degree of HE, nor between GEC and blood ammonia concentration. Cirrhosis patients without HE had the highest initial clearance of I3N-ammonia and cirrhosis patients with HE the lowest initial clearance. AUCs of 13N-ammonia was significantly higher in patients with HE compared to the other two groups, showing a decreased ammonia blood clearance. Total I3N-radioactivity concentration in healthy controls and patients without HE ended at similar levels, but at a higher level in patients with HE. Arterial pH in HE patients was elevated. Lower pH in patients without HE could make clearance of ammonia more dependent on regulated membrane transport proteins and partly explain why some cirrhosis patients develop symptoms of HE. High turnover of ammonia to glutamine in muscles might reduce the risk of developing HE and increased deamination of glutamine might increase the risk of HE. This is supported by the finding of lower late clearance of I3N-ammonia in patients with HE compared to patients without HE and healthy controls but no significant difference in the early clearance. Conclusions: Patients with liver cirrhosis and HE clear blood ammonia much slower than patients with cirrhosis but no HE and healthy controls. Our data support other findings that pH and muscular glutamine production seem to influence development of HE.
12091 EX VlVO ENDOTOXIN REMOVAL FROM PLASMA RESTORES NEUTROPHIL FUNCTION
V Stadlbauer, R.P. Mookerjee, S. Lidder, S.J. Hodges, N.A. Davies, R. Williams, R. Jalan. The 7JCL Institute ~ f ’ H e p t o l o g yLondon, , UK E-mail: [email protected] Background: In patients with alcoholic hepatitis, organ failure and mortality are often related to infection suggesting a state of ‘immune failure’. We previously demonstrated that neutrophils from patients with alcoholic hepatitis are fully activated (high resting burst) and show decreased phagocytic capacity. High resting burst and decreased phagocytosis was associated with infection, organ failure and mortality. Therefore we aimed to investigate whether endotoxin can cause these changes in neutrophils and whether endotoxin removal from patients’ plasma can restore neutrophil function ex vivo. Method: Peripheral blood neutrophils were isolated from patients or healthy volunteers and incubated with endotoxin-spiked plasma from controls or plasma from patients. Neutrophil oxidative burst activity and phagocytic capacity was assayed by FACS analysis using the Phagoburst test and Phagotest (Orpegen). Endotoxin was removed either by passing the plasma over a polymixin B containing column (Detoxi-Gel, Pierce Biotechnology) or incubation with an LPS-neutralizing anti-CD14 antibody (Clone llD18) Results: Incubating neutrophil from healthy volunteers with increasing endotoxin concentrations (50-200ng/ml) resulted in a concentrationdependent increase in resting burst (n = 5 , p < 0.0001). Incubating patients’ neutrophils with endotoxin reduced phagocytosis by 20% (n = 8, p 1 0.05). When plasma was passed over Detoxi-Gel columns, resting burst was reduced by 32% (p 0.00 I , n = 9) and phagocytosis increased by 3 1 % (p 1 0.05, n = 1 I ) . When plasma from patients with low resting burst (n = 8) and normal plasma ( n = 5 ) was passed over the column, no changes in resting burst or phagocytosis were observed. Incubation with an anti-CD 14 antibody prevented the induction of increased burst in normal neutrophils by high burst patient plasma (p < 0.001, n = 7 ) and increased phagocytosis
S87
by 20% (p 0.05, n = 1 I ) , whilst having no effect on neutrophils incubated with low burst (n = 8) or healthy control plasma (n = 3). Discussion: Our data support the notion that endotoxin may cause functional changes in neutrophils similar to those observed in patients with alcoholic hepatitis and that ex vivo removal of endotoxin restores neutrophil function. This novel observation may have important therapeutic implications: Patients with high resting burst and decreased phagocytic capacity, who are at increased risk for infection, might benefit from endotoxin removal strategies rather than from treatment with steroids.
12101 EVIDENCE FOR A PATHOGENETIC ROLE OF ASYMMETRIC DIMETHYLARGININE (ADMA) IN CIRRHOTIC PORTAL HYPERTENS1ON F. Vizzutti’, R.G. Romanelli’, U. Arena’, L. Rega2, M. Brogi’, F. Marra’, M. Foschi’, R. Tarquini’, G. Laffi’, M. Pinzani’. ‘Dipurtimrnto di Mrdicinu Internu Uniuersitu di Firrnzr, Firenzr; 2Dipurtimmto di Rudiologiu, Aziendu Osprduliero Uniuer~xituriuCureggi Firrnzr, Firrnze, Italy E-mail: [email protected]
Background: Chronic liver diseases (CLD) are frequently complicated by portal hypertension, an important component of which is the increased intrahepatic vascular resistance, in part related to intra-hepatic endothelial dysfunction. ADMA, an endogenous inhibitor of NO synthase (NOS), has emerged in a variety of cardiovascular disease as a mediator of endothelial dysfunction at relatively low plasma concentrations. Aim: Although elevated ADMA plasma level have been reported in endstage CLD, there is no clear evidence of ADMA involvement in the pathogenesis of portal hypertension in humans. We therefore investigated the possible implication of ADMA in this clinical setting. Patients and Methods: We studied 39 consecutive patients with HCV related CLD at different stage of fibrotic evolution: 34 cirrhotic, and 5 non-cirrhotic patients (METAVIR F2 to F3). All patients underwent HVPG measurements, and simultaneous blood samples were taken from the hepatic vein and pulmonary artery, for ADMA and NOx plasma levels determinations. Exclusion criteria were: cardiopulmonary disease, arterial hypertension, diabetes mellitus, hypercholesterolemia, hyperomocysteinemia, acute and chronic renal failure, chronic inflammatory diseases, recent active alcohol intake, use of vasoactive drugs andor NSAIDs. Results: A positive correlation among HVPG and ADMA concentrations in hepatic veins (ADMA-h) and pulmonary artery (ADMA-p) (r=0.77, p iO.OOOO1 and r=0.56, p < 0.00001, respectively) was found. Moreover, a negative correlation between HVPG and NOx concentrations in hepatic vein (NO-h) (r=-0.50, p <0.005), and between ADMA-h and NO-h was observed (r=-0.40, p < 0.02). ADMA-p levels (0.55f0.13 LimoliL) were significantly higher than ADMA-h ones (0.47&0.09 pmol/L) (p < 0.00001). Systemic and hepatic vein ADMA and NOx concentrations did not correlate either with Child-Pugh class or score in cirrhotic patients. However, when patients were classified according to Child-Pugh score 1 7 and 28, a correlation with ADMA-p levels was 0.02). observed (p i Conclusions: ADMA-h is directly related to portal pressure and inversely related to NO-h, thus indicating that ADMA may play a pathophysiological role contributing to the relative intra-hepatic NO deficiency typical of the so-called cirrhotic endothelial dysfunction.