1. In Vitro Intestinal Absorption
Baldeo, Biendima, Go, Olivar, Soriano

2. 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

3. 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 mL
bicarbonate buffered frog ringer’s
Measure Absorbance at 540 nm of Filtrate
solution + 250 mg glucose +/- DNP

4. Rationale
• Buffered Ringer’s  protect surface of
intestine
• Aeration  provide oxygen
• Eversion of the intestine  exposure of
absorptive surfaces

5. 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.

6. 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%

7. Glucose Transport
enzymes are located in the enterocytes
covering the intestinal microvilli brush border

8. 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

10. 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

11. Generation of ATP
• Electron transport chain – H+ gradient – ATP
synthase

12. 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

14. 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

15. Filtrates from
Solution A (w/o DNPH) Solution B (w/ DNPH)
Absorbance HIGH LOW
Excess/Unreacted HIGH LOW
Benedict’s
Glucose in Filtrate LOW HIGH
Glucose absorbed by HIGH LOW
intestinal segments

16. 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, 25mL Ringer’s Solution,
250mg Glucose, frog 250mg Glucose, frog
intestine intestine, dinitrophenol
Initial color Blue Green
Final color Green Red
Conclusion Relatively low amount of Relatively high amount of
glucose indicates normal glucose in the solution
intestinal absorption. indicates inhibition of
intestinal absorption.