Figure 1.
The two major pathways metabolizing ammonia: the urea cycle (periportal), and glutamine synthesis (pericentral).
In the urea cycle: ammonia and bicarbonate form carbamoylphosphate via carbamoyl phosphate synthetase1 (CPS1). This reaction requires N-acetylglutamate (acquired via a reaction catalysed by N-acetylglutamate synthase (NAGS)), Mg2+ and MgATP. Carbamoylphosphate combines with ornithine in a reaction catalysed by ornithine carbamoyltransferase (OTC) to form citrulline. Citrulline is transported to the cytosol and combines with aspartate to form argininosuccinate (reaction catalysed by argininosuccinate synthetase (ASS1)). Argininosuccinate is then cleaved by argininosuccinate lyase (ASL) yielding fumarate and arginine. Arginase (ARG1) cleaves arginine, producing urea and ornithine. Urea is excreted as waste and ornithine is transported back to the mitochondria to be used in subsequent cycles of urea synthesis. In the pericentral hepatocytes, ammonia ‘escaping’ the urea cycle is metabolized to glutamine (reaction catalysed by glutamate–ammonia ligase (GLUL).
Table 1.
Primers used for qPCR.
Table 2.
Antibody characteristics, manufacturers, dilutions and protocol specifications for immunohistochemistry.
Figure 2.
The ten most significantly enriched signalling pathways in dogs with EHPSS.
MetaCore pathway analysis based on gene expression differences between healthy dogs and dogs with EHPSS in the microarray analysis.
Table 3.
mRNA expression differences of ammonia metabolizing enzymes detected in the microarray.
Figure 3.
Relative mRNA expression of ammonia metabolizing enzymes in liver tissue of control and CPSS dogs.
Significant differences between mRNA expression of the enzymes in control liver samples compared to samples obtained intra-operatively (intra) are indicated by stars (* = p<0.05, ** = p<0.01). There were no significant differences in relative mRNA expression of the enzymes in the liver samples obtained intra-operatively and two months post-operatively (post) in dogs achieving complete recovery.
Figure 4.
Immunohistochemical localization of ammonia metabolizing enzymes in liver of healthy dogs.
GLUL (a) was exclusively expressed immunohistochemically around the central veins (C) whereas CPS1 (b), OTC (c) and ASS1 (d) were localized around the portal vessels (P). ASL (e) and ARG1 (f) lacked a zonal distribution pattern.
Figure 5.
Immunohistochemical localization of CPS1, OTC and ASS1 in liver of dogs with CPSS.
In contrast to the periportal distribution of CPS1 in healthy dogs, CPS1 (a+b) showed a slightly more intense staining around the central vein in one dog with an EHPSS (a) and both around the pericentral and periportal vessels in three dogs with an IHPSS (b). No immunoreactivity for OTC was seen in seven dogs with CPSS (c). ASS1 (d) was not distributed according to a zonal pattern as opposed to the periportal distribution seen in the healthy control dogs. In one dog a more intense staining was seen in both periportal and the pericentral areas. C, central vein. P, portal vein.
Figure 6.
Effect of ammonia on primary hepatocytes.
Cell number (n = 3) and urea production (n = 4) in primary hepatocytes after treatment with different ammonia concentrations, data depicts average with standard deviation (A). Increased ammonia production and unchanged cell numbers (indicated by CyQUANT) indicate vitality of hepatocytes and active UCE. Relative expression of urea cycle enzymes in primary hepatocytes (control n = 4; treatment n = 2) (B). Primary hepatocytes were treated with either 0, 6, 60, 600 or 6,000 µM ammonia for 3 hours.