experimental setup for histidine transport and estimation of histidine using pauly's reaction

Sodium-Dependent Histidine Absorption in Goat Jejunal Inverted Sacs

Biological cells are surrounded by a membrane lipid bilayer that separates the intracellular cytosol from the extracellular content. The membrane serves as a semipermeable barrier that regulates the movement of solutes1. Transport across biological membranes is affected by a solute&#39;s permeability coefficient, which depends on several factors, including the concentration and charge of the solute. In general, solutes move through the membrane using three mechanisms (Figure 1): Passive diffusion, facilitated diffusion, and active transport2. Simple diffusion is the process by which soluble, uncharged, and non-polar solutes pass down their concentration gradient through a semipermeable membrane (Figure 1A). Membrane proteins do not assist in this process since it involves the movement of solutes from a region of higher concentration to a region of lower concentration. The rate of diffusion is based on Fick&#39;s Law3. On the other hand, facilitated diffusion is a protein-dependent transport wherein the membrane allows only selective solutes to pass along a concentration gradient without the expenditure of energy (Figure 1B). This kind of transport is specific and differs from simple diffusion in exhibiting saturation kinetics.
Active transport is a protein-dependent transport of molecules against their concentration gradient, i.e., from the region of lower concentration to a region of higher concentration with the use of ATP or ion gradients (Figure 1C). When the transporter hydrolyses ATP, the transport is termed primary active transport (Figure 1C; left panel). Another form of active transport is secondary active transport (Figure 1C; right panel). In secondary active transport, solutes are moved based on an electrochemical gradient. It takes place when a transporter protein couples the movement of an ion (typically Na+) down its concentration gradient with the movement of another molecule or an ion against its concentration gradient. This kind of solute movement can be a co-transport (Symport) wherein both the solute and the ion move in the same direction or an exchange (Antiport), in which case the solute and the ion move in opposite directions.
The dietary amino acids and monosaccharides from food sources are absorbed in the small intestine. The small intestine can be functionally divided into three segments: Duodenum, Jejunum, and Ileum (Figure 2). Absorption of solute occurs throughout the small intestine, with maximum absorption occurring at the jejunum and at the proximal end of the ileum. The intestinal enterocytes are polarized cells, and the tight junctions connecting two adjacent cells create two distinct membrane sites - the basolateral and the apical membrane site (Figure 2). The absorption of luminal solutes generated by digestion occurs on the apical membrane site4.
Histidine transport in the intestinal enterocytes at the apical membrane is an example of a secondary active symport that is sodium-dependent. At the basolateral end, the histidine entering the enterocyte moves down the concentration gradient into the hepatic portal circulation. The intracellular concentrations of sodium within the enterocyte are maintained at 12 mmoles/L1, which is lower than the extracellular/luminal concentrations, due to the active pumping of sodium out of the cell by Na+ K+ATPase located on the basolateral membrane (Figure 3). At the apical membrane of enterocytes, the B0AT and the sodium neutral amino acid transporter (SNAT) 5 are the major transporters that not only transport histidine but also amino acids such as asparagine and glutamine in a sodium-dependent cotransport5,6. Another transport protein called Large Amino acid Transporter (LAT)1 present at the basolateral membrane of enterocytes transports large neutral amino acids such as leucine, tryptophan, tyrosine, and phenylalanine across the membrane7.
With the objective of teaching the concept of membrane transport integrated with techniques such as spectrophotometry and routine biochemical assays through experiential learning pedagogy, it is imperative to develop methodologies that can not only demonstrate the concept in easy-to-understand terms but also enable participatory learning for undergraduate students. Currently, there are limited resources available to the students for hands-on engagement to learn such concepts of biochemistry. Here, we report a simple protocol for demonstrating Histidine transport across goat intestinal membrane that is easy to reproduce in undergraduate laboratories and can be adapted for evaluating the transport of other metabolites as well. More importantly, the method utilizes inexpensive materials in an undergraduate laboratory, thereby enabling experiential learning in even the simplest of laboratory settings.

Author Spotlight: Experiential Tool for Teaching Active Transport Using Ex Vivo Histidine Uptake

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 the, 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 dissections.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 memory and 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 X vivo 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.

Experimental Setup for Histidine Transport and Estimation of Histidine Using Pauly's Reaction

To begin, assemble two sets of 350-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 microcentrifuge 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 Pauly's reaction followed Lambert-Beer's law til 300 micromolars of histidine.A correlation was observed between the uptake of histidine and sodium concentrations in jejunal enterocytes.

experimental-setup-for-histidine-transport-estimation-histidine-using

Research

JoVE Journal

Biochemistry

Histidine is an essential amino acid that is also a precursor for metabolites implicated in the immune system, pulmonary ventilation, and vascular circulation. Absorption of dietary histidine relies largely on the sodium-coupled neutral amino acid transport by the Broad neutral amino acid transporter (B0AT) present on the apical membrane of the enterocyte. Here, we demonstrate the absorption of histidine by the intestinal villus enterocytes from the lumen using goat jejunal inverted sacs. The jejunal sacs exposed to varying concentrations of sodium and histidine were assayed to determine the concentration of histidine inside the sacs as a function of time. The results show active histidine absorption. Increasing the concentration of salt resulted in higher absorption of histidine, suggesting a symport of sodium and histidine absorption in goat intestinal inverted sacs. This protocol may be applied to visualize the intestinal mobility of amino acids or other metabolites with appropriate modifications. We propose this experiment as an experiential pedagogical tool that can help undergraduate students comprehend the concept of membrane transport.

Here, we report an inexpensive and reproducible method demonstrating the membrane transport of histidine in a goat intestine. This process occurs by co-transporting histidine and sodium ions enabled by the sodium gradient across the enterocyte membrane. This method exploits experiential learning pedagogy to better understand solute movement across biological membranes.

Histidine is an essential amino acid that is also a precursor for metabolites implicated in the immune system, pulmonary ventilation, and vascular ...

Preparation of Inverted Jejunum Sacs for Demonstrating the Membrane Transport of Histidine

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 sacs 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.