PT - JOURNAL ARTICLE AU - Haeyoung Zhang AU - Chris Wolford AU - Abdul Basit AU - Albert P Li AU - Peter W Fan AU - Ryan H Takahashi AU - S. Cyrus Khojasteh AU - Bill J Smith AU - Bernard P Murray AU - Kenneth Thummel AU - Bhagwat Prasad TI - Regional proteomic quantification of clinically relevant non-cytochrome P450 enzymes along the human small intestine AID - 10.1124/dmd.120.090738 DP - 2020 Jan 01 TA - Drug Metabolism and Disposition PG - dmd.120.090738 4099 - http://dmd.aspetjournals.org/content/early/2020/04/29/dmd.120.090738.short 4100 - http://dmd.aspetjournals.org/content/early/2020/04/29/dmd.120.090738.full AB - Current challenges in accurately predicting intestinal metabolism arise from the complex nature of the intestine, leading to limited applicability of available in vitro tools, as well as knowledge deficits in intestinal physiology, including enzyme abundance. In particular, information on regional enzyme abundance along the small intestine is lacking, especially for non-cytochrome P450 (non-CYP) enzymes such as carboxylesterases (CES), UDP-glucuronosyltransferases (UGTs), and sulfotransferases (SULTs). We used cryopreserved human intestinal mucosa (CHIM) samples from nine donors as an in vitro surrogate model for the small intestine and performed LC-MS/MS-based quantitative proteomics for 17 non-CYP enzymes, using stable isotope-labeled peptides. Relative protein quantification was done by normalization with enterocyte marker proteins, i.e., villin-1, sucrase isomaltase, and fatty acid binding protein 2, and absolute protein quantification is reported as pmol per mg protein. Activity assays in glucuronidations and sequential metabolisms were conducted to validate the proteomics findings. Relative or absolute quantifications are reported for CES1, CES2, five UGTs, and four SULTs along the small intestine: duodenum, jejunum, and ileum for six donors and in ten segments along the entire small intestine (A - J) for three donors. Relative quantification using marker proteins may be beneficial in further controlling for technical variabilities. Absolute quantification data will allow for scaling factor generation and in vivo extrapolation of intestinal clearance using physiologically-based pharmacokinetic (PBPK) modeling.SIGNIFICANCE STATEMENT Current knowledge gaps exist in intestinal protein abundance of non-cytochrome P450 (non-CYP) enzymes. Here, we employ quantitative proteomics to measure non-CYP enzymes along the human small intestine in 9 donors, using cryopreserved human intestinal mucosa (CHIM) samples. Absolute protein abundances are reported, as well as relative protein abundance normalized with enterocyte markers. Corresponding activities are also examined.