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Unlabelled: Ammonia metabolism in the liver has been largely credited to hepatocytes (HCs). We have shown that liver nonparenchymal cells that include liver sinusoidal endothelial cells (LSECs) produce ammonia. To address the limited knowledge regarding a role for LSECs in ammonia metabolism, we investigated the ammonia metabolism of isolated LSECs and HCs under three different conditions: (1) bioreactors containing LSECs (LSEC-bioreactors), (2) bioreactors containing HCs (HC-bioreactors), and (3) separate bioreactors containing LSECs and HCs connected in sequence (Seq-bioreactors). Our results showed that LSEC-bioreactors released six-fold more ammonia (22.2 nM/hour/10(6) cells) into the growth media than HC-bioreactors (3.3 nM/hour/10(6) cells) and Seq-bioreactors (3.8 nM/hour/10(6) cells). The glutamate released by LSEC-bioreactors (32.0 nM/hour/10(6) cells) was over four-fold larger than that released by HC-bioreactors and Seq-bioreactors (<7 nM/hour/10(6) cells). LSEC-bioreactors and HC-bioreactors consumed large amounts of glutamine (>25 nM/hour/10(6) cells). Glutaminase is known for catalyzing glutamine into glutamate and ammonia. To determine if this mechanism may be responsible for the large levels of glutamate and ammonia found in LSEC-bioreactors, immunolabeling of glutaminase and messenger RNA expression were tested. Our results demonstrated that glutaminase was present with colocalization of an LSEC-specific functional probe in lysosomes of LSECs. Furthermore, using a nucleotide sequence specific for kidney-type glutaminase, reverse-transcription polymerase chain reaction revealed that this isoform of glutaminase was expressed in porcine LSECs., Conclusion: LSECs released large amounts of ammonia, perhaps due to the presence of glutaminase in lysosomes. The ammonia and glutamate released by LSECs in Seq-bioreactors were used by hepatocytes, suggesting an intrahepatic collaboration between these two cell types.

Unlabelled: Treatment of hyperammonemia and hepatic encephalopathy in cirrhosis is an unmet clinical need. The aims of this study were to determine whether L-ornithine and phenylacetate/phenylbutyrate (administered as the pro-drug phenylbutyrate) (OP) combined are synergistic and produce sustained reduction in ammonia by L-ornithine acting as a substrate for glutamine synthesis, thereby detoxifying ammonia, and the phenylacetate excreting the ornithine-derived glutamine as phenylacetylglutamine in the urine. Sprague-Dawley rats were studied 4 weeks after bile duct ligation (BDL) or sham operation. Study 1: Three hours before termination, an internal carotid sampling catheter was inserted, and intraperitoneal saline (placebo), OP, phenylbutyrate, or L-ornithine were administered after randomization. BDL was associated with significantly higher arterial ammonia and brain water and lower brain myoinositol (P < 0.01, respectively), compared with sham-operated controls, which was significantly improved in the OP-treated animals; arterial ammonia (P < 0.001), brain water (P < 0.05), brain myoinositol (P < 0.001), and urinary phenylacetylglutamine (P < 0.01). Individually, L-ornithine or phenylbutyrate were similar to the BDL group. In study 2, BDL rats were randomized to saline or OP administered intraperitoneally for 6 hours or 3, 5, or 10 days and were sacrificed between 4.5 and 5 weeks. The results showed that the administration of OP was associated with sustained reduction in arterial ammonia (P < 0.01) and brain water (P < 0.01) and markedly increased arterial glutamine (P < 0.01) and urinary excretion of phenylacetylglutamine (P < 0.01) in each of the OP treated groups., Conclusion: The results of this study provide proof of the concept that L-ornithine and phenylbutyrate/phenylacetate act synergistically to produce sustained improvement in arterial ammonia, its brain metabolism, and brain water in cirrhotic rats.

Unlabelled: Hyperammonemia is a feature of acute liver failure (ALF), which is associated with increased intracranial pressure (ICP) and brain herniation. We hypothesized that a combination of L-ornithine and phenylacetate (OP) would synergistically reduce toxic levels of ammonia by (1) L-ornithine increasing glutamine production (ammonia removal) through muscle glutamine synthetase and (2) phenylacetate conjugating with the ornithine-derived glutamine to form phenylacetylglutamine, which is excreted into the urine. The aims of this study were to determine the effect of OP on arterial and extracellular brain ammonia concentrations as well as ICP in pigs with ALF (induced by liver devascularization). ALF pigs were treated with OP (L-ornithine 0.07 g/kg/hour intravenously; phenylbutyrate, prodrug for phenylacetate; 0.05 g/kg/hour intraduodenally) for 8 hours following ALF induction. ICP was monitored throughout, and arterial and extracellular brain ammonia were measured along with phenylacetylglutamine in the urine. Compared with ALF + saline pigs, treatment with OP significantly attenuated concentrations of arterial ammonia (589.6 +/- 56.7 versus 365.2 +/- 60.4 mumol/L [mean +/- SEM], P= 0.002) and extracellular brain ammonia (P= 0.01). The ALF-induced increase in ICP was prevented in ALF + OP-treated pigs (18.3 +/- 1.3 mmHg in ALF + saline versus 10.3 +/- 1.1 mmHg in ALF + OP-treated pigs;P= 0.001). The value of ICP significantly correlated with the concentration of extracellular brain ammonia (r(2) = 0.36,P< 0.001). Urine phenylacetylglutamine levels increased to 4.9 +/- 0.6 micromol/L in ALF + OP-treated pigs versus 0.5 +/- 0.04 micromol/L in ALF + saline-treated pigs (P< 0.001)., Conclusion: L-Ornithine and phenylacetate act synergistically to successfully attenuate increases in arterial ammonia, which is accompanied by a significant decrease in extracellular brain ammonia and prevention of intracranial hypertension in pigs with ALF.