[13C] phenylalanine breath test and hepatic phenylalanine metabolism enzymes in cirrhotic rats

Background Stable isotope C-13-labelled phenylalanine breath test has been applied to enable the quantitative evaluation of hepatic functional reserve, but the mechanism underlying the changes in function has not been resolved. This study evaluated the correlation between expression of the mRNA of key enzymes mediating phenylalanine metabolism and the metabolism of L-[1-C-13] phenylalanine (C-13-phe) assessed by the excretion of C-13-CO2 in the breath of rats with, and without, chronic hepatic injury induced by administration of carbon tetrachloride (CCl4). Materials and methods Male Sprague-Dawley (SD) rats (n = 29) were given subcutaneous injections of CCl4 to induce chronic hepatic injury. L-[1-C-13] phenylalanine breath tests (PheBT) were then applied to the rats to assess hepatic function. Expression of phenylalanine hydroxylase (PHH) and tyrosine transaminase (TYT) mRNA in liver was detected by real-time fluorescence quantification RT-PCR, using TaqMan as the probe. It was then determined whether the PheBT results correlated with PHH and/or TYT mRNA expression. In addition, immunohistochemical labelling was used to visualize PHH protein expression in the control and injured liver tissue. Results There were significant decreases in PheBT and PHH mRNA expression in the cirrhotic rats relative to the uninjured controls and these two measures of liver function were correlated. However, TYT mRNA expression was not changed by CCl4-induced liver injury. The immunohistochemical analysis revealed that PHH protein was expressed predominantly in the cytoplasm of liver cells. Conclusions The results of the PheBT were consistent with the changes in PHH gene expression following liver injury. The present findings indicate that decreased expression of the rate-limiting enzyme PHH, but not of TYT, might underlie the functional deficits detected as decreased PheBT. The C-13 excretion rate constant per mass liver (PheBT-k/LW) was the most sensitive index that could be used to evaluate the PHH mRNA expression in the liver.