Franck Montigon

Development of a rapid and convenient method to purify mucins and determine their in vivo synthesis rate in rats.

The intestinal mucoprotein synthesis rate was measured in vivo for the first time. For this, a rapid, reproducible, and convenient method to purify mucoproteins from large numbers of intestinal samples at the same time was developed. The method takes advantage of both the high mucin resistance to protease activities due to their extensive glycosylations and the high mucin molecular size. Intestinal homogenates were partially digested with Flavourzyme. Nonprotected proteins partially degraded were easily separated from mucoproteins by small gel filtration chromatography using Sepharose CL-4B. Electrophoretically pure mucins were obtained. Their amino acid composition was typical of purified intestinal epithelial mucins. The mucoprotein synthesis rate was determined in vivo in rats using the flooding dose method with the stable isotope L-[1-13C]valine. Free L-[1-13C]valine enrichments in the intracellular pool were determined by GC-MS. L-[1-13C]valine enrichments into purified mucoproteins or intestinal mucosal proteins were measured by gas chromatography-combustion-isotope ratio mass spectrometry. In rats, we found that the gut mucosa protein synthesis rate (%/day) decreased regularly from duodenum (122%/day) to colon (43%/day). In contrast, mucoprotein fractional synthesis rates were in the same range along the digestive tract, between 112%/day (colon) and 138%/day (ileum).

Mucin Production and Composition Is Altered in Dextran Sulfate Sodium-Induced Colitis in Rats

We evaluated the small and large intestinal mucin production in a rat model of human ulcerative colitis by measuring the in vivo fractional synthesis rate (FSR) and the expression of mucins. A chronic colitis was induced by oral administration of 5% dextran sulfate sodium (DSS) for 9 days followed by 2% DSS for 18 days. DSS-treated rats showed increased colonic MUC2,3 mRNA levels compared pair-fed controls. The mucin FSR was unaffected while mucin-containing goblet cells were depleted in the vicinity of lesions. In the small intestine, no inflammatory lesions were observed but ileal MUC2 mRNA levels and mucin FSR were decreased by 46% and 21%, respectively. Finally, DSS-treated rats showed a marked decrease in mucins threonine + serine content all along the gut, which may lead to a reduction of potential O-glycosylation sites. Our data indicate that the chronic colitis may impair the mucus layer protective function all along the gut.

Determination of 13C- and 15N-enrichment of glutamine by gas chromatography/mass spectrometry and gas chromatography/combustion/isotope ratio mass spectrometry after N (O, S)-ethoxycarbonyl ethyl ester derivatisation

To measure 13C- and 15N-Gln enrichments in plasma samples by gas chromatography/mass spectrometry (GC/MS) and gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) we investigated the use of the N(O,S)-ethoxycarbonyl ethyl ester derivative. We found that this derivative is very stable at room temperature, even after 5 days storage. It allows reproducible and accurate 13C- and 15N-isotopic enrichment determinations with values of RSD below 0.8 and 3.2% for GC/MS and GC/C/IRMS, respectively. This derivative enables high sample throughput analyses when automated GC/MS and GC/C/IRMS are used. The methodology was applied to measure isotopic enrichments in rat plasma after oral force-feeding with [2,5-15N2]-Gln and in human plasma samples obtained after intravenous infusion of [1-13C]-Gln. In rats after oral force-feeding, we found that the maximal appearance of [2,5-15N2]-Gln occurred between 40 and 80 min in the two compartments studied (portal and peripheral blood). In humans infused intravenously with [1-13C]-Gln for 4 h, the plateau in 13C-Gln enrichments in plasma was reached after 3 h of infusion and coefficients of variation between samples were 1.7 and 12.4% for the two subjects. Using the N(O,S)-ethoxycarbonyl ethyl ester derivative, 13C- and 15N-isotopic enrichment can be determined in the range 2–100 ± 0.3 MPE by GC/MS and 0.3–2 ± 0.02 MPE by GC/C/IRMS. Copyright

The Chronic Colitis Developed by HLA-B27 Transgenic Rats Is Associated with Altered In Vivo Mucin Synthesis

HLA-B27 transgenic rats spontaneously developing a chronic inflammation mainly involving the colon are recognized as a powerful animal model for IBD. We investigated the mucin production in 6-month-old HLA-B27 rats by measuring in vivo fractional synthesis rate (FSR) and expression of mucins. In the inflamed colon of HLA-B27 rats, the mucin FSR was stimulated by 75% compared to F-344 controls, while MUC2,3 mRNA expression was unchanged. A local depletion in mucus-containing goblet cells was observed, suggesting a rapid mucin production/release and/or a real global decrease in goblet cell number. In the noninflamed jejunum of HLA-B27 rats, the mucin FSR was reduced by 35% compared to controls, while MUC2,3 mRNA expression was unchanged. Different alterations in mucin metabolism and expression are observed between HLA-B27 rats and a model of chemically induced chronic colitis (DSS-treated rats), suggesting that mucin alterations may be dependent on the animal model and colitis underlying mechanism.