Short communication: Transport of 2-hydroxy-4-methyl-thio-butanoic isopropyl ester by rumen epithelium in vitro

Our objective was to evaluate the potential of rumen epithelium to transport 2-hydroxy-4-(methylthio)-butanoic isopropyl ester (HMBi) using the Ussing chamber technique. Rumen tissues were obtained from a nearby slaughterhouse, separated from the muscle and serosal layer as quickly as possible after exsanguination, placed in buffer, and gassed with 95:5 (vol/vol) O2:CO2 before tissue mounting. Two levels of HMBi (0.44 and 0.88mg/mL) and 2 incubation times (120 and 180min) were used in 12 chambers with 3 replicates per treatment with an exposed surface area of 2cm2. Four separate experiments were conducted (n=16). Concentrations of HMBi and methionine hydroxy analog (HMB) were measured by HPLC in rumen-side and serosal-side buffers. Data are expressed as percentage of added HMBi. Initial time samples were taken for comparison with incubated samples. Adding the HMBi-buffer mixture to the rumen side caused an immediate release of HMB (mean=6.3%). Breakdown of HMBi to HMB at initial time was due to hydrolysis reactions at the epithelial surface. Overall, a small and variable amount of HMBi was transferred to the serosal buffer (mean of 0.58% across both times and both concentrations). A larger amount of HMB (8.94%) was isolated in the serosal buffer. Increasing incubation time increased the amount of HMB in the ruminal buffer (34.0% at 120min vs. 43.4% at 180min) and decreased the amount of HMBi (37.9% at 120min vs. 28.1% at 180min). These data indicate that very limited amounts of HMBi may cross the rumen epithelium. The amount of HMB isolated on the serosal side was about 10 times higher than HMBi. The hydrolysis of HMBi to HMB required the presence of rumen tissue or perhaps microbes attached to the tissue. Based on this in vitro system, direct transport from the rumen would not explain rapid blood methionine increases observed when HMBi is fed.