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In Vitro Intestinal AbsorptionBaldeo, Biendima, Go, Olivar, Soriano
Methodology: Preparation of Intestinal Segments Bathe Intestine with warm Turn the buffered Cut everted Remove intestinal Pith Frog Ringer’s intestinal rings Intestine segment inside solution while and strips out continuously aerating it
Methodology: Experiment Proper Draw 0.5 mL sample at 20-min intervals for 2 hours Test tube: sample + 4 mL Benedict’s Regent Heat for 3 minutes Cool and filter Incubating Medium: 25 mLbicarbonate buffered frog ringer’s Measure Absorbance at 540 nm of Filtratesolution + 250 mg glucose +/- DNP
Rationale• Buffered Ringer’s protect surface of intestine• Aeration provide oxygen• Eversion of the intestine exposure of absorptive surfaces
Benedict’s Reagent• Reducing sugars are oxidized by the copper ion in solution to form a carboxylic acid and a reddish precipitate of copper (I) oxide.
Benedict’s Test• Test for the presence of reducing sugars (aldehydes and alpha- hydroxyketones)• Reduction of Cu2+ to Cu+ ions (precipitated as CuO)• Green - 0.5%, Yellow – 1%, Orange – 1.5%, Red - >2%
Glucose Transport enzymes are located in the enterocytescovering the intestinal microvilli brush border
Glucose Transport• glucose absorption occurs in a co-transport mode with active transport of sodium• initial active transport of sodium through the basolateral membranes of the intestinal epithelial cells that provides the eventual motive force for moving glucose also through the membranes
Hexoses vs. Pentoses• fructose is not co-transported with sodium, its overall rate of transport is only about one half that of glucose or galactose• Fructose phosphorylation glucose• Fructose transporter GLUT5 - passive
Generation of ATP• Electron transport chain – H+ gradient – ATP synthase
Dinitrophenol• At low pH, the basic form acquires an H+ and converts to the acidic form.• At high pH, the acidic form gives up its H+ to convert to the basic form.• Uncouples oxidation of compounds to generation of ATP
Other inhibitors• Phlorhizin – glycoside that displaces sodium from its binding site. As a result, glucose could not be bound and transported.• Oubain – Na+ pump inhibitor• excess K+ or Li+ – Na+ pump inhibitor• Flavonoids – GLUT2 transporter
Filtrates from Solution A (w/o DNPH) Solution B (w/ DNPH) Absorbance HIGH LOW Excess/Unreacted HIGH LOW Benedict’s Glucose in Filtrate LOW HIGHGlucose absorbed by HIGH LOW intestinal segments
Table 1. The result showed decrease in absorption of glucose in solution Bdue to the inhibitor (dinitrophenol) present compared to solution A based on the estimated quantified unabsorbed glucose in the solution determined using Benedict’s test. Solution A Solution BComponents 25mL Ringer’s Solution, 25mL Ringer’s Solution, 250mg Glucose, frog 250mg Glucose, frog intestine intestine, dinitrophenolInitial color Blue GreenFinal color Green RedConclusion Relatively low amount of Relatively high amount of glucose indicates normal glucose in the solution intestinal absorption. indicates inhibition of intestinal absorption.