Study On Biological Mechanism And Clinical Application Of L-[1-～(13)C] Phenlalanine Breath Test For Assessment Of Liver Function

Various 13CO2 breath tests have been used for quantitative assessment on liver function involving microsomal, cytosolic and mitochondrial function, which is safe, simple, non-invasive, instantaneous, holistic. The elementary research of these tests has been ongoing for years. Three of main reasons may be the potential pharmacological side effects of the test substrate, possible inductivity of exogenous substances and drugs to interrelated enzymes involving substrate catabolism and undefined biological mechanism of these breath tests. L-[1-13C]phenylalanine breath test (PheBT) has been reported by several researchers to quantify liver function of some kinds of subjects. Most of their tests show PheBT is valuable in diagnosing hepatopathy.Objective: To establish a effective model for animal breath test in experimental research, study the activity of phenylalanine hydroxylase (PAH) in the liver of rats and the correlation between the results of PheBT and the PAH activity, study the expression of PAH and tyrosine aminotransferase (TYT) mRNA in rat liver and correlation with the results of PheBT, study the expression of PAH protein and local the protein in the liver of the rats, investigate the biological mechanism of PheBT and the feasibility and validity of using the parameters of PheBT to reflect liver function and injure of hepatocytes in rat liver, investigate the feasibility of oral PheBT in evaluating liver function of patients with hepatitis B virus-related liver cirrhosis in comparison with the conventional liver blood tests and Child-Pugh score, optimize the methodology of clinical PheBT, and proposal effective parameters for quantitative assessment of liver function.Methods:Acute hepatitis was induced by CCl4 administration through an intragastric gavage to Prague-Dawley rats. PheBT was assisted by small ventilator improved and air samples were collected discontinuously, 20mg/kg body weight L-[1-13C]phenylalanine (13C-phe) was administered intravenously. The parameter 13C excretion rate constant (PheBT-k) was applied to quantify hepatic phenylalanine metabolism and liver function.PAH activity in liver tissue was determined by enzyme-coupling rate procedure. The correlation between the activity of phenylalanine hydroxylase of acute hepatitis and cirrhosis rat liver and results of PheBT was investigated.Cirrhosis was induced by administered CCl4 subcutaneously on rats. Intravenous PheBT was performed. The parameters PheBT-k and PheBT-k per liver weight (PheBT-k/LW) were used to liver functional reserve and hepacytote injure.The liver tissue was stained with both hematoxylin-eosin and picrosirius; The severity of liver disease based on the score of necrosis and inflammatory reaction and fibrosis was assessed in liver biopsy specimens by a skillful pathologist and by image analysis system.Fluorescence quantitative polymerase chain reaction (real time quantitative polymerase chain reaction), FQ-PCR, based on Taq Man technology has been established to detect the mRNA expression of PAH and TYT and to identify the relationship between PheBT-k/LW and their expression.PAH protein expression and its distribution in liver tissue were studied by immunohistochemistry.Various dosages (50mg, 100mg and 150mg/body) of oral 13C-phe and different fasting period (0.5, 2, 4, 7, 12h) before oral administration of 13C-phe were investigated to develop a superior PheBT for diagnosis of liver disease. The results of oral PheBT were investigated between different gender and age (<40 and >40) in healthy adult subjects.PheBT was performed after 12 hours fasting and oral administration of 100mg/body of 13C-phe to determined the difference among healthy controls and the three cirrhotic groups according to Child-Pugh classification, and compared the result of PheBT with standard liver blood tests and Child-Pugh classification score.Results:The total PAH activity of the whole liver in acute CCl4 hepatitis rats and PheBT-k were significantly decreased compared to control rats; The parameter PheBT-k was...