Persistence of sugars used for intestinal permeability measures in an in vitro rumen environment

Orally administered synthetic sugars are routinely used as markers of intestinal permeability in nonruminants and young calves, but not adult ruminants, likely because of uncertainty surrounding degradation of such sugar markers (e.g., d-mannitol, sucralose, lactulose) in the rumen. The objective was to evaluate persistence of d-mannitol, sucralose, and lactulose in a closed in vitro rumen fermentation system over 48 h. The null hypothesis was that sugar concentration would not be affected by time. Rumen contents were collected and processed under anerobic conditions a total of 12 times from a ruminally cannulated lactating Holstein cow. These 12 rumen samplings reflect 4 in vitro experiments (d-mannitol, sucralose, lactulose, and d-glucose as a positive methodological control), each replicated 3 times. For each replication, filtered rumen contents and rumen buffer (1:3; vol/vol) were added to a series of six 500-mL flasks, each containing 3 filter bags. Each filter bag contained 500 mg of ground total mixed ration (94.2% dry matter; 15.2% crude protein, 40.9% neutral detergent fiber, 3.9% fat, and 6.2% ash, dry matter basis) and three 5-mm glass beads. The 6 flasks represented 0, 6, 12, 24, and 48 h time points, and a 48-h negative control flask. A single sugar was tested during each experimental replicate. Final flask concentrations of each sugar were 4.07 mg/mL d-glucose, 1.99 mg/mL d-mannitol, 2.17 mg/mL sucralose, or 3.10 mg/mL lactulose. Flasks were incubated under anerobic conditions at 39°C where they remained undisturbed until the designated time of removal (0, 6, 12, 24, or 48 h). At removal, an aliquot of each flask was removed and sugar concentration was quantified by HPLC-mass spectrometry. Data for each experiment were analyzed using an ANOVA model that included the single fixed effect of time (0, 6, 12, 24, or 48 h); flask within replicate was the random term. Lactulose was not resolved in any samples due to interfering components within the sample matrix; no lactulose data are presented. As expected, positive methodological control of glucose decreased to negligible concentrations by 6 h of in vitro incubation. d-Mannitol followed the same pattern as glucose, which was different from our hypothesis. The interpretation is that d-mannitol is degraded in the in vitro rumen culture system and, by extension, is therefore not a viable choice to use in in vivo intestinal permeability tests in adult ruminants when dosed orally. As hypothesized, sucralose concentration did not change over 48 h of incubation in a closed in vitro rumen fermentation system. This suggests feasibility of orally dosed sucralose in adult ruminants as a rumen-inert marker of intestinal permeability with subsequent analysis of biological samples (e.g., urine, blood) by HPLC-mass spectrometry.