Refined methodology to purify mucins from pig colonic mucosa

Libao-Mercado, A. J.; Lange, Catherine

doi:10.1016/j.livsci.2007.01.125

Cited by 13 publications

(16 citation statements)

References 6 publications

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“…Determination of the quantitative utilization of threonine by the gastrointestinal tract was not possible in humans, so we used the minipig as an animal model for adult humans. In this model, as previously observed for calves (19) and pigs (20), threonine accounted for 10% of the total amino acids in intestinal mucins. This contribution was much lower than that reported in humans (2).…”

Section: Discussionsupporting

confidence: 68%

Intestinal Inflammation Increases Gastrointestinal Threonine Uptake and Mucin Synthesis in Enterally Fed Minipigs

Rémond

Buffière

Godin

et al. 2009

The Journal of Nutrition

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The high requirement of the gut for threonine has often been ascribed to the synthesis of mucins, secreted threonine-rich glycoproteins protecting the intestinal epithelium from injury. This requirement could be even greater during intestinal inflammation, when mucin synthesis is enhanced. In this study, we used an animal model to investigate the effects of an acute ileitis on threonine splanchnic fluxes. Eight adult multi-catheterized minipigs were fed with an enteral solution. Four of them were subjected to experimental ileitis involving direct administration of trinitrobenzene sulfonic acid (TNBS) into the ileum (TNBS-treated group) and the other 4 were not treated (control group). Threonine fluxes across the portal-drained viscera (PDV) were quantified with the use of simultaneous i.g. L-[(15)N]threonine and i.v. L-[U-(13)C]threonine infusions. Ileal mucosa was sampled for mucin fractional synthesis rate measurement, which was greater in the TNBS-treated group (114 +/- 15%/d) than in the control group (61 +/- 8%/d) (P = 0.021). The first-pass extraction of dietary threonine by the PDV and liver did not differ between groups and accounted for approximately 27 and 10% of the intragastric delivery, respectively. PDV uptake of arterial threonine increased from 25 +/- 14 micromol x kg(-1) x h(-1) in the control group to 171 +/- 35 micromol x kg(-1) x h(-1) in the TNBS-treated group (P < 0.001). In conclusion, ileitis increased intestinal mucin synthesis and PDV utilization of threonine from arterial but not luminal supply. This leads to the mobilization of endogenous proteins to meet the increased threonine demand associated with acute intestinal inflammation.

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Section: Discussionsupporting

confidence: 68%

Intestinal Inflammation Increases Gastrointestinal Threonine Uptake and Mucin Synthesis in Enterally Fed Minipigs

Rémond

Buffière

Godin

et al. 2009

The Journal of Nutrition

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“…Ice-cold EDTA buffer (5 mM) was added to mucus samples so as to yield a final concentration of 150 mg mucus/ml EDTA and were subsequently homogenized according to Libao-Mercado and De Lange (2007). After homogenization, the samples were Tsirtsikos, Fegeros, Kominakis, Balaskas and Mountzouris centrifuged at 10 000 3 g for 30 min at 48C and the soluble mucin-containing supernatant was collected.…”

Section: Sampling and Processingmentioning

confidence: 99%

“…After homogenization, the samples were Tsirtsikos, Fegeros, Kominakis, Balaskas and Mountzouris centrifuged at 10 000 3 g for 30 min at 48C and the soluble mucin-containing supernatant was collected. The purification of mucin was performed by size-exclusion chromatography using an XK-16 column (GE Healthcare Bio Sciences AB, Uppsala, Sweden) filled in with Sepharose CL-4B (SigmaAldrich, St. Louis, MO, USA) and equilibrated with the elusion buffer (0.2 M NaCl), according to Libao-Mercado and De Lange (2007). The high molecular mucin molecule was eluted at the void volume of the column and collected as 1 ml fractions detected via combined UV (280 nm) and refractive index detection.…”

Section: Sampling and Processingmentioning

confidence: 99%

Modulation of intestinal mucin composition and mucosal morphology by dietary phytogenic inclusion level in broilers

Tsirtsikos

Fegeros

Kominakis

et al. 2012

Animal

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The effect of a dietary phytogenic feed additive (PFA) inclusion level in mucin monosaccharide composition, mucosal morphometry and mucus histochemistry along the broiler intestinal tract was studied. Cobb male broilers (n 5 525) were allocated into five experimental treatments that, depending on the type of addition in the basal diet (BD), were labeled as follows: C (BD based on maize-soybean meal with no other additions), E1 (80 mg PFA/kg BD), E2 (125 mg PFA/kg BD), E3 (250 mg PFA/kg of BD) and A (2.5 mg avilamycin/kg BD). Samples from duodenum, ileum and cecum of 14-and 42-day-old broilers were collected and analyzed. In 14-day-old broilers, treatments E2 and E3 had higher (P , 0.01) duodenal mannose than treatments C, E1 and A. Ileal mannose was lower (P , 0.05) in treatment C compared with PFA treatments, and ileal galactose (Gal) was higher (P , 0.01) in treatments E2 and E3 compared with C and A. Polynomial contrast analysis with respect to PFA inclusion level showed that in 14-day-old broilers there was a linear increase (P 5 0.001) in duodenal mannose and a quadratic effect (P 5 0.038) in duodenal N-acetyl-galactosamine with increasing PFA level. Ileal Gal and mannose increased linearly (P 5 0.002 and P 5 0.012, respectively) with PFA inclusion level. There were no significant differences between treatments in mucin monosaccharide molar ratios of 42-day-old broilers. However, increasing PFA inclusion level resulted in a linear decrease of ileal fucose (P 5 0.021) and cecal N-acetylgalactosamine (P 5 0.036). Experimental treatments did not differ (P . 0.05) regarding duodenal villus height (Vh), crypt depth (Cd) and Vh/Cd ratio, irrespective of broiler age and the intestinal segment examined. However, increasing dietary PFA inclusion level showed a pattern of linear increase of duodenal Vh/Cd ratio in 14-day-old broilers and ileal Vh in 42-day-old broilers (P 5 0.039 and P 5 0.039, respectively). Alcian Blue-Periodic AcidSchiff (pH 2.5) staining of neutral and acidic mucins showed that the staining intensity of mucus layer in villi was fragment (i.e. tip, midsection and base) dependent, whereas in crypts it was dependent both on intestinal segment (i.e. duodenum, ileum and cecum) and fragment. Finally, mucus layer thickness did not differ (P . 0.05) between treatments, yet a pattern of linear increase (P , 0.05) with PFA inclusion level was observed in the duodenum of 42-day-old broilers. In conclusion, the dietary inclusion level of PFA modulated broiler intestinal mucin composition and morphology. Further studies are required to elucidate the physiological implications of such changes in host-microflora interactions.Keywords: phytogenic feed additive, essential oils, broiler, mucin, intestinal morphology ImplicationsThis study has shown that the inclusion level of a phytogenic feed additive in broiler diets has resulted in changes in the gut mucin monosaccharide composition and the intestinal mucosal architecture morphology. Given the fact that animal performance and disease prevention are prerequisite...

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“…8 However, because of inter-and intramolecular disulde bonds, mucins are linked together to form polymers of up to 40 MDa. [9][10][11] 80% of the glycoprotein's molecular weight is assigned to carbohydrate moieties that make mucin mostly hydrophilic and play a major role in the protein's net negative charge and its functionality. Hydrophobic patches within the protein backbone contribute to the excellent adsorption onto hydrophobic surfaces 12 and serve as the basis for the high lubricity of mucins.…”

Section: Introductionmentioning

confidence: 99%

“…While older publications mainly focused on the analysis of mucin structure and provided only small amounts of protein, 9,[22][23][24] no studies concentrated on high yields and productivities. In our former studies, a robust reference process (see Fig.…”

Section: Introductionmentioning

confidence: 99%

Solid liquid liquid extraction of porcine gastric mucins from homogenized animal material

et al. 2017

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Mucins purified from porcine stomachs have gained importance in biomedical applications as they exhibit unique features such as hydrogel formation, lubricity and antivirality. Because commercially available porcine gastric mucins (PGM) are neither gel forming, nor do they reduce friction, a robust purification process for functional mucins from porcine tissue (mainly Muc5AC) is necessary. Based on our former investigations (V. J. Schömig, B. T. Käsdorf, C. Scholz, K. Bidmon, O. Lieleg and S. Berensmeier, RSC Adv., 2016, 6, 44932-44943), we further optimized the established purification process in terms of productivity and overall yield as well as ease of scalability. We therefore introduced a novel extraction method -solid liquid liquid extraction (SLLE) -as an early capture step from homogenized porcine stomach, which combines conventional solid liquid extraction with a second, immiscible solvent, to simultaneously delipidate the tissue and extract hydrophilic proteins into the polar phase. Using Design of Experiments (DoE) the parameters incubation time and ratio of solvent phases (hexane/water) were identified to 3 h and 1/15 for SLLE, respectively. PGM was collected in the polar phase and further purified by size exclusion chromatography and diafiltration. With the homogenization of porcine stomach and the introduction of the SLLE, up to 3570 mg mucin per stomach can be purified, resulting in 55 times more PGM compared to the former published reference process. The productivity of the process was up to 25 mg mucin per stomach and per h, representing an improved process productivity by a factor of 4. Lubricity and gel formation of the purified mucin were retained with the optimized extraction protocol.

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Refined methodology to purify mucins from pig colonic mucosa

Cited by 13 publications

References 6 publications

Intestinal Inflammation Increases Gastrointestinal Threonine Uptake and Mucin Synthesis in Enterally Fed Minipigs

Intestinal Inflammation Increases Gastrointestinal Threonine Uptake and Mucin Synthesis in Enterally Fed Minipigs

Modulation of intestinal mucin composition and mucosal morphology by dietary phytogenic inclusion level in broilers

Solid liquid liquid extraction of porcine gastric mucins from homogenized animal material

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