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Methodology: Preparation of Intestinal Segments
Pith FrogRemove Intestine
Bathe Intestine with warm buffered Ringer’s
solution while continuously
aerating it
Turn the intestinal
segment inside out
Cut evertedintestinal rings
and strips
Methodology: Experiment Proper
Incubating Medium: 25 mL bicarbonate buffered frog ringer’s solution + 250 mg glucose +/- DNP
Measure Absorbance at 540 nm of Filtrate
Cool and filter
Heat for 3 minutes
Test tube: sample + 4 mL Benedict’s Regent
Draw 0.5 mL sample at 20-min intervals for 2 hours
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 enterocytes covering 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
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
Benedict’s
HIGH LOW
Glucose in Filtrate LOW HIGH
Glucose absorbed by
intestinal segments
HIGH LOW
Table 1. The result showed decrease in absorption of glucose in solution B due 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 B
Components 25mL Ringer’s Solution, 250mg Glucose, frog intestine
25mL Ringer’s Solution, 250mg Glucose, frog intestine, dinitrophenol
Initial color Blue Green
Final color Green Red
Conclusion Relatively low amount of glucose indicates normal intestinal absorption.
Relatively high amount of glucose in the solution indicates inhibition of intestinal absorption.