| Liver is the major ogran of ammonia metabolism. The gastrointestinal original ammonia is normally removed by the liver via portal vein and entered the urea cycle for irreversible conversion to urea. The ammonia concentration in the portal vein of pig is up to 10-fold greater than elsewhere in the circulation. And the preliminary study showed that with the increase of dietary protein level, the concentration of portal ammonia is reached 250μmol/L-350μmol/L. The aim of this study was to explore the effects of portal ammonia on the hepatic amino acid metabolism in pig. Firstly, pigs were surgically implanted with catheter in the portal vein and hepatic vein to establish portal infusion technique. Then high concentration（75mmol/L） and low concentration（25mmol/L） of ammonium chloride（NH4Cl） were infused into liver by portal catheter. Serum samples were collected to characterize metablites based on gas chromatography-mass spectrometry（GC-MS） metabolomics. Liver samples were collected to examine gene expression by digital gene expression profiles and related metabolic enzyme activity. Finally, mechnism of the influence of 15N labeled NH4 Cl on hepatocyte amino acid metabolism was investigated. The main results were as follows:（1） Establishment of portal infusion technique in liver of pig. The portal blood ammonia flow（μmol/min） was calculated based on the portal blood velocity and the concentration of portal ammonia before and after feeding. The concentration of ammonia in perfusion was calculated accdording to the infusion rate of pump in vitro. Then pigs, fitted acutely with transhepatic catheters, were infused with low or high concentration of NH4 Cl. The urea concentraion of plasma was detected in hepatic vein. It was found that with the increase of ammonia concentration in perfusate, the urea synthesis was increased. This results confirmed the reliability of portal infusion technique.（2） The results of GC-MS analysis showed that: there were 12 different metabolites in serum between two concentration of NH4 Cl infusion（P<0.05）; in contrast with low concentration of NH4 Cl, the infusion of high ammona concentration resulted in the significant synthesis of urea and glutamine（P<0.05） together with the glucose and glucose-6-phosphate; on the contrary, the content of alanine（Ala）, aspartate（Asp）, glutamate, ornithine and arginine were remarkably decrease（P<0.05）, among which Ala was the greatest reduction.（3） The results of digital gene expression profiles analysis showed that: compared with low concentration, there were 1164 differentially expressed genes（DEGs） in high concentration treament, in which 901 DEGs were up-regulated and 263 DEGs were down-regulated（P<0.05）; in this DEGs, the gene related urea cycle such as Nacetylglutamate synthase, carbamoyl-phosphate synthase 1, ornithine carbamoyltransferase and arginase 1 were significant up-regulated（P<0.05）; the gene related amino acid metabolism were also changed（P<0.05） such as glutamine synthetase, ornithine aminotransferase; the results of quantitative PCR were in accordance with Ion proton sequencing result.（4） The results of metablic enzyme activity in liver showed that: the hepatic enzyme activity of carbamoyl-phosphate synthase 1, arginase 1, glutamine synthetase in high concentration of NH4 Cl were higher than that in low concentration（P<0.05）; however, the activity of ornithine carbamoyltransferase（P=0.054） and glutaminase（P=0.065） were tended to increase; with the increase of NH4 Cl concentration infusion, the activity of alanine aminotransferase（ALT） and aspartate aminotrasferase（AST） were increased 45.89%（P=0.039） and 41.08%（P=0.045）, respectively. These results indicated that ammonia lead to increase the activity of ALT and AST.（5） The results of 15N enrichment in urea and Asp based on GC-MS analysis showed that: there was much massive production of 15N-urea（m+1）（urea with one nitrogen atom labeled） and less production of 15N-aspartate when only incubate with 15NH4 Cl. However, when 15N-Ala and NH4 Cl as the substrate, there was largely one nitrogen atom labeled in urea and the 15N enrichment in Asp was almost 4 times（P<0.05） in that 15NH4 Cl. Both nitrogen of urea was largely labeled when hepatocyte incubate in 15N-Ala plus 15NH4 Cl. Compared with 15N-Ala+NH4Cl group, no obvious changes was observed（P>0.05） in 15N-Ala+15NH4Cl with regard to 15N enrichment in Asp, but the 15N enrichment in Urea（m+1） was significant reduction（P<0.05）. These results indicated that ammonia had an inability to provided additional nitrogen for urea synthesis, and Ala was mainly through generating Asp for providing precursor to urea cycle.Taken together: portal ammonia enter urea cycle and can only provide one nitrogen for urea in liver. The additional nitrogen source is supplyed mainly through amino acid metabolism. Thus, the metabolism of ammonia via urea cycle in liver could induce Ala metabolism, following transamination to Asp via ALT and AST for providing the additional nitrogen for urea. |
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