ABSTRACT
The HepatoPac micropatterned coculture (MPCC) hepatocyte system has been shown to be an effective tool to investigate the qualitative human and preclinical species' metabolite profiles of new drug candidates. However, additional improvements to the overall study conditions and execution, layout, and human-donor count could be made. To that end, we have evaluated several ways to increase the amount of data one can generate per MPCC plate and how to more efficiently execute a MPCC study for the purpose of metabolite generation. Herein, we compare a set of compounds using single- and 10-donor pooled human MPCC hepatocytes. Intrinsic clearance and mean metabolic activities assessed by diverse enzyme markers were comparable between the single- and 10-donor pool. We have confirmed that the generated metabolite profiles were indistinguishable between the single- and 10-donor pool and also that rat MPCC can be performed at 400 µl media volume, which greatly simplifies study execution. Additional tips for successful study execution are also described. SIGNIFICANCE STATEMENT: When using the HepatoPac micropatterned coculture (MPCC) system, sometimes simple experimental condition variables or problematic plate designs can hamper productive study execution. We evaluated conditions to increase the amount of data one can generate per MPCC plate and, perhaps more importantly, execute that study more efficiently with less likelihood of error. We describe some of our key learnings, provide an examination of enzyme activity levels and clearance values, and provide some recommendations to simplify the execution of a HepatoPac experiment.
Subject(s)
Hepatobiliary Elimination , Metabolomics/methods , Primary Cell Culture/methods , Animals , Biotransformation , Chromatography, High Pressure Liquid/methods , Coculture Techniques/methods , Datasets as Topic , Drug Evaluation, Preclinical/methods , Female , Fibroblasts , Hepatocytes/metabolism , Humans , Male , Rats , Tandem Mass Spectrometry/methodsABSTRACT
Five crossbred beef steers (329 kg) were used in a 5 x 5 Latin square experiment with 14-d periods to determine the effects of supplementation with high-nitrogen (N) feeds alone or mixed with tallow on sites of digestion with a basal diet of bermudagrass hay. Hay was 1.93% nitrogen, 75% neutral detergent fibre and fed at 1.83% of body weight (dry matter; DM). Supplements were basal (B; 105 g DM): 81.8% dried molasses product (DMP) and 18.2% calcium carbonate (CC); soybean meal (S; 942 g DM): 88.0% soybean meal, 9.8% DMP and 2.2% CC; S mixed with 9.8% tallow (SF; 1041 g DM); corn gluten and blood meals (CB; 662 g DM): 62.5% corn gluten meal, 20.8% blood meal, 13.6% DMP and 3.0% CC; CB mixed with 13.2% tallow (CBF; 757 g DM). Total N intake was 117, 185, 187, 174 and 172 g/d, and duodenal N flow was 121, 148, 143, 162 and 169 g/d for B, S, SF, CB and CBF, respectively, being lower for B than for other treatments and higher for supplements with the corn gluten and blood meal mix than for soybean meal (P less than 0.05). Duodenal microbial N flow was 39, 51, 49, 38 and 45 g/d for B, S, SF, CB and CBF, respectively, being greater (P less than 0.05) for supplements with soybean meal than with corn gluten and blood meals. Duodenal flow of feed N was greater (P less than 0.05) with than without high-N feeds and for supplemental corn gluten and blood meals than for soybean meal (78, 90, 86, 117 and 116 g/d for B, S, SF, CB and CBF, respectively). In conclusion, mixing of tallow and high-N feeds did not affect the extent of ruminal N disappearance, and soybean meal supplementation increased duodenal N flow less than did supplementation with corn gluten and blood meals. Increased duodenal N flow with soybean meal was associated with about equal elevations of ruminal outflow of microbial and feed N, whereas the corn gluten-blood meal mix affected the intestinal protein supply by increasing ruminal escape of feed protein.
