4/16/2023 0 Comments Liver focus![]() The remaining tissue samples were used for the isolation of hepatocytes and subsequent global proteomic analysis. These samples were used for global proteomic analysis of liver tissue as described below. A total of 12 of the tissue samples were snap-frozen immediately after excision and stored at −80 ☌. Ethical approval was granted by the Uppsala Regional Ethics Committee (ethical approval no. Samples of normal human liver tissue were obtained from 20 donors undergoing surgical resections carried out at the Department of Surgery, Uppsala University Hospital in Uppsala, Sweden. We propose a simple approach for extrapolating in vitro data to predict in vivo outcome. Using the protein abundance data, we show that primary hepatocytes are likely to underpredict the in vivo CL of pitavastatin and other drugs that are dependent on transporters for their hepatic elimination. (16, 17) Moreover, altered protein expression was associated with the protein ubiquitination pathway, which may explain the reduced levels of plasma membrane proteins. Pathway analysis of those proteins for which expression levels differed significantly support previous observations that the hepatocytes are exposed to oxidative stress during isolation. A lower fraction of mitochondrial membrane proteins in the isolated hepatocytes was also observed. In contrast, the levels of hepatocyte-specific drug transporters and many other proteins present in the plasma membrane were lower in the primary cells compared to those in liver tissue. (14, 15) Our analysis revealed that proteins in the endoplasmic reticulum membrane, including many hepatocyte-specific drug metabolizing cytochrome P450 (CYP) enzymes, were retained during isolation and sample preparation. Freshly isolated hepatocytes were selected for the analysis to distinguish isolation-related changes from culture-dependent proteome changes. Herein, we performed in-depth, quantitative LC–MS/MS analysis of the membrane proteome of human liver tissue and freshly isolated hepatocytes, with a focus on proteins that determine hepatic drug exposure. (8, 9) Advantages of the label-free approach include the large number of proteins that are quantified simultaneously and the dynamic range of detection. (7) As a result, accurate determinations of protein levels from label-free LC–MS/MS analysis are now feasible using the total protein approach. Recent advances in instrumentation and sample preparation have improved the sensitivity and throughput of liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis to a level that allows almost complete proteome coverage. Furthermore, important ADME proteins, such as transport proteins in the plasma membrane, are often expressed in low amounts, making their detection and quantification difficult. Most ADME proteins are integral membrane proteins, which complicates their analysis. This is partly related to technical issues. Nevertheless, there are few quantitative global expression analyses of proteins that determine hepatic exposure. Knowledge of both protein abundance and function is key for understanding drug metabolism and transporter-mediated distribution. We tentatively conclude that our data set will be a useful resource for improved hepatocyte predictions of the in vivo outcome. ![]() Finally, using pitavastatin as an example, we show how protein quantifications can improve in vitro predictions of in vivo liver clearance. Pathway analysis of the differentially expressed proteins confirmed that hepatocytes are exposed to oxidative stress during isolation and suggested that plasma membrane proteins were degraded via the protein ubiquitination pathway. This included transport proteins that determine hepatocyte exposure to many drugs and endogenous compounds. ![]() However, the expression of many plasma membrane proteins was lower in the isolated hepatocytes than in the liver tissue. There was a good global correlation in protein abundance. A total of 5144 unique proteins were identified, spanning over 6 orders of magnitude in abundance. Here, we quantified and compared the membrane proteomes of freshly isolated hepatocytes and human liver tissue using a label-free shotgun proteomics approach. For accurate predictions of the in vivo outcome, the isolated hepatocytes should reflect the phenotype of their in vivo counterpart, i.e., hepatocytes in human liver tissue. Freshly isolated human hepatocytes are considered the gold standard for in vitro studies of liver functions, including drug transport, metabolism, and toxicity.
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