In vitro intestinal absorption

In Vitro Intestinal Absorption

Baldeo, Biendima, Go, Olivar, Soriano

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

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

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.