Demonstration of Membrane Transport of Histidine using Goat Intestinal Inverted Sacs: An Experiential Pedagogical Tool for Undergraduates

Here we propose a pedagogical tool for undergraduate students of biology where we are demonstrating the concept of membrane transport. What we are actually doing here is with the objective of showing the transport of histidine, which is an amino acid across the enterocyte membrane in a simple, easy to perform, inexpensive experimental setting, which can be done in almost any laboratory. The current challenge that we face in an undergraduate laboratory is the ban that has been imposed on animal dissects.

Now, because of this, what we are doing here is procuring the intestines from a common vendor. And intestines that we do get, however, have a problem because they are not from syngeneic animals. And therefore, there would be a variability in the animals samples that we get.

So currently, there are no simple and easy-to-perform experiments that can facilitate learning about the concepts of membrane transport. Most of the methods available use hazardous chemicals that are not feasible for teaching learning in a UG setting. So this technique demonstrates an XVIVO setup that doesn't rely on cell-free extracts, but a tissue sample which can be derived from different available mammalian sources, so that's the obvious advantage to our method.

To begin, place the goat entrails in PBS so that they are immersed completely for cleaning. Separate the small intestine from the large intestine. Using scissors, remove the external connective tissue.

Gently flush the small intestine with PBS using a 10 milliliter syringe to remove undigested food material and obtain a hollow bag of small intestine. Using a ruler, measure the complete length of the small intestine. Divide the intestine into three parts, duodenum, and the remaining part equally into jejunum and ileum.

Employing a blade, remove all connective tissue to clean the jejunum. Cut the jejunum into 12 to 15 centimeter-long portions. After washing the portions with PBS, place them in a Petri dish containing PBS.

Using a glass rod, invert the jejunum portions to expose the Villi on the outside. Gently flush the inverted jejunum portions with PBS. Check for any leaks from the sides of the inverted jejunum.

Then cut the inverted jejunum portions into five centimeter-long sacs. Tie one end of each sac with twine. Fill the sac with PBS to recheck for visual leaks.

Tie the other end of the sacs to create empty inverted sacs with villi on the external surface. Pat the inverted sac on filter paper to recheck for external leaks. To begin, assemble two sets of three 50 milliliter tubes with different concentrations of sodium chloride.

Dip the prepared intestinal sacs in the specified tubes for 30 minutes and 60 minutes for each set. After the designated time, empty the solution inside the sac in a separate 1.5 milliliter micro centrifuge tube, and determine the volume. Using a two milliliter syringe, aspirate the liquid inside the sac.

To create a standard curve for histidine, prepare solutions of varying histidine concentrations using histidine stock solutions and water. Then add sulfanilic acid and sodium nitrate to each tube. After incubating the tubes for five minutes at room temperature, add one milliliter of sodium carbonate and ethanol to each tube.

Finally, measure the absorbance at 490 nanometer wavelength. Histidine estimation using poly's reaction followed Lambert-Beer's law till 300 micromolars of histidine. A correlation was observed between the uptake of histamine and sodium concentrations in jejunal enterocytes.