Reduced clearance of proteins labeled with diisopropylfluorophosphate in portacaval-shunted rats

Portacaval shunting is a model for hepatic encephalopathy that causes chronic hyperammonemia, disruption of metabolic, signaling, and neurotransmitter systems, and progressive morphological changes. Exposure of cultured cells to ammonia raises intralysosomal pH and inhibits proteolysis, and the present study tested the hypothesis that proteolytic capacity is diminished in portacaval-shunted rats. Proteins were labeled in vivo with tracer doses of diisopropylfluorophosphate (DFP) and clearance of label was assayed. This approach labeled proteins independent of protein synthesis, which is reported to be altered in shunted rats, and avoided complications arising from re-utilization of labeled amino acids that causes underestimation of degradation rate. Characterization of DFP labeling showed that protein labeling was fast, about 50 % of the label was released during a 24 h interval, labeling by DFP metabolites was negligible, inhibition of brain acetylcholinesterase was not detectable, and labeling by [³H]- and [¹⁴C]DFP was equivalent. To assay degradative capacity, proteins were first labeled with [³H]DFP, followed by labeling with [¹⁴C]DFP that was given 24 or 72 h later. The ³H/¹⁴C ratio in each animal was used as a relative measure of removal of ³H-labeled proteins. ³H/¹⁴C ratios were generally significantly higher in portacaval-shunted rats than in controls, consistent with reduced proteolytic capacity. Assays of amino acid incorporation into brain protein generally replicated literature reports, supporting the conclusion that protein synthesis unlikely to be markedly inhibited and amino acid recycling influences calculated protein synthesis rates in shunted rats. Therapeutic strategies to reduce ammonia level would help normalize lysosomal functions and protein and lipid turnover.