Digestion in the Small Intestines

EXPERIMENT 13
DIGESTION IN THE SMALL INTESTINES

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ABSTRACT
The small intestine serves as the site of major digestive and absorptive processes. In this experiment, the action of pancreatic enzymes on representative samples of each food group under different conditions, such as increased/decreased pH and presence of other substances, were observed. A pancreatin solution was first prepared from a hog pancreas and was completely neutralized using 0.5% Na2CO3. Afterwards, 3 mL each of the neutral pancreatin solution was placed in 7 test tubes and were further added with other specified reagents. The test tubes containing HCl were not effective in digesting the biomolecules since pancreatin is only active in moderately alkaline conditions, as observed in test tube 2. Data obtained from test tubes 3-7 were inconclusive and would most likely subjected to errors, thus, producing anomalous results.

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DISCUSSION OF DATA AND RESULTS The small intestine is divided into three segments, namely, (1) duodenum which originates from the pyloric sphincter of the stomach and extends for 25 cm, (2) jejunum that is approximately 2.5 m long, and (3) ileum which is 3.6 m long and joins the large intestine at the ileocecal sphincter. (Tortora & Anagnostakos, 1990)
Major portions of digestion and absorption occur in the small intestine for three reasons: (1) food entering the intestine have undergone extensive preliminary breakdown due to the mechanical and chemical processes in the mouth and stomach; (2) the small intestine receives enzymatic secretions which are necessary in the complete digestion of each major food group; and (3) food stays in the small intestine for a relatively long period of time which allows the enzymes to completely act upon the biomolecules. (Chaffee & Greisheimer, 1975)
Pancreatin is a combination of pancreatic enzymes, namely, amylase, lipase, and protease—each individual component act on starch, lipids, and proteins, respectively. The solution was prepared from a hog pancreas which was washed with sodium hydroxide (NaOH) and neutralized with 0.5% sodium carbonate (Na2CO3). Pancreatin and other intestinal enzymes are active at moderately alkaline pH as compared to stomach enzymes which require acidic conditions for them to be active. (Indiana University, 2010)
Table 1. Summary of results for the action of pancreatic enzymes TEST TUBE # | RESULTS | 1 | Hard-boiled egg white: Digestion occurred | 2 | Hard-boiled egg white: No digestion | 3 | The color of iodine disappears. | 4 | The color of iodine disappears. | 5 | Titration: 2 drops of NaOH used (overtitrated) | 6 | Titration: 1 drop of NaOH used | 7 | Titration: 1 drop of NaOH used |

In the experiment, equal slices of hard-boiled egg whites were dropped into test tubes 1 and 2, which were both containing neutral pancreatin, and 0.5% Na2CO3 and 0.01N hydrochloric acid (HCl), respectively. From these combinations of reagents, it is clear that test tube 1 would contain a basic solution while test tube 2 would contain an acidic one. Observations showed that digestion occurred in test tube 1 while no digestion was seen in test tube 2.
The rate at which digestion occurs is affected by various factors, but more importantly by the dependency of the digestive processes on enzyme activity. Enzymes require certain optimal conditions for them to be able to act on specific substances effectively and efficiently. Temperature and pH are two factors that greatly affect enzymatic activity and reactions. The rate of enzyme-catalyzed reactions generally increases when temperature is increased, provided that the temperature increase is within the range at which the enzyme does not denaturize and retains full activity; on the other hand, most enzymes would have a characteristic pH at which their activity is maximal and a decrease or increase of the pH would result to a decline in activity. (Lehninger, 1970) Thus, pancreatin, in particular the protease, would require a moderately alkaline condition for it to be active and digest the protein in the hard-boiled egg white. The alkaline condition is met in test tube 1 and is expected to show some sign of digestion, which was indeed observed. Although, protease does not completely break down the protein into its constituent amino acids, it fragments the proteins further and intestinal amino acids would eventually complete the digestion. On the other hand, the acidic condition in test tube 2 would inactivate the pancreatin, along with the other intestinal enzymes, thus no digestion would occur. In actual digestion, the hormone secretin causes the pancreas to secrete a thin, watery juice that is rich in Na2CO3 which helps in neutralizing the HCl in the chyme (the form in which food enters the small intestine from the stomach). (Chaffee & Greisheimer, 1975) The duodenal mucosa is not equipped to withstand the corrosive effects of gastric juice, thus, neutralization not only preserves the activity of the enzymes, but also protects the duodenal mucosa. Starch solution, representing carbohydrates, was added to the neutral pancreatin in test tubes 3 and 4. Sodium carbonate was added to test tube 3, while HCl and bile salts were added to test tube 4. Similar to the digestion of the protein in egg white, the requirement for the enzyme amylase to be active is for it to be present in moderately alkaline condition. The Na2CO3 added to test tube 3 fulfills this condition. The presence of HCl and bile salts in test tube 4 does not help in the digestion of starch into maltose, because (1) HCl deactivates the pancreatin and (2) bile salts don’t act upon carbohydrates but on fats. Thus, digestion is more expected to happen in test tube 3 than in test tube 4. However, results show the same observations for both test tubes. Vegetable oil (neutral) was added to the neutral pancreatin contained in test tube 5, 6, and 7. Calcium chloride was also added to test tubes 5 and 6. Distilled water was added to test 6 and 7. Bile salts were further added to test tube 6. The results obtained for this part of the experiment were rather inconclusive, since the titration data are not comparative. For test tube 6 and 7, it required only 1 drop of the NaOH for it to turn into faint pink. On the other hand, the solution in test tube 5 was already over titrated after 2 drops of NaOH were added. Accordingly, fats are acted upon by pancreatic lipase (which is in the neutral pancreatin solution) and are split into glycerol and fatty acids, but its action is more effective when the fats are emulsified first by the action of bile salts. (Chaffee & Greisheimer, 1975) Moreover, studies show that CaCl2 can “jumpstart” the hydrolysis action of pancreatic lipase on fats and shifts the pH of pancreatin by one unit down. (Schonheyder & Volqvartz, 2008) Thus, it is to be expected that the test tube which would most likely be able to recreate this environment would have the greatest amount of digestion. The titration volume would be serving as an indicative of the amount of digestion that occurred in each of the test tubes. The greater the volume used in titration, the greater the amount of digestion that occurred, inciting a directly proportional relationship.
Thus, comparing the test tube contents, test tube 6 would most probably have the greatest amount of digestion; since it contains pancreatin, CaCl2, and bile salts, whose functions have already been discussed in preceding paragraphs. Basically, the presence of the aforementioned substances would recreate or imitate the condition in the intestine thus allow better digestion as compared to the other test tubes. The vegetable oil would be more digested in test tube 6 since the neutral pancreatin’s pH would be shifted to a lower pH (more alkaline) due to the action of CaCl2, apart from the fact that it activates the pancreatic lipase to hydrolyze the fat. Furthermore, bile salts would emulsify the fats first so that the digestion would be more effective. The other test tubes (test tubes 5 and 7) don’t contain CaCl2 which was supposed to help activate the pancreatic lipase, thus, digestion of the fat would be impossible due to the fact that the enzyme is inactive. Pancreatic and intestinal enzymes require moderately alkaline conditions for them to be active and perform their specific functions in the digestive process. When the optimal conditions are not met, the process would most probably not occur.
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REFERENCES
Campbell, M. K., & Farrell, S. O. (2009). Biochemistry . California: Thomson Brooks/Cole.
Chaffee, E. E., & Greisheimer, E. M. (1975). Basic Physiology and Anatomy. Quezon City: MW Publishing, Inc.
Garrett, R. H., & Grisham, C. M. (2010). Biochemistry. Massachusetts: Brooks/Cole.
Indiana University. (2010). Retrieved March 8, 2012, from Indiana University: http://www.indiana.edu/~nimsmsf/P215/p215notes/LabManual/Lab12.pdf
Lehninger, A. L. (1970). Biochemistry. New York: Worth Publishers, Inc.
Schonheyder, F., & Volqvartz, K. (2008). On the Activation of Pancreatic Lipase by Calcium Chloride at Varying pH. Acta Physiologica Scandinavica , 62-69.
Tortora, G. J., & Anagnostakos, N. P. (1990). Principles of Anatomy and Physiology. New York: Harper & Row, Publishers.

ANSWERS TO QUESTIONS 1. What are the end products of the action of pancreatic amylase on starch?

Pancreatic amylase would hydrolyze starch into a disaccharide, in particular, maltose.

2. Why was hard-boiled egg and not raw egg white used in this experiment?

3. How can you make use of the Biuret reagent to follow the progress of the tryptic digestion of a solid protein under optimum conditions?

4. What is the role of CaCl2 in the action of pancreatic lipase?

Calcium chloride (CaCl2) activates the hydrolysis action of pancreatic lipase on triglycerides and would lower the pH of the pancreatic lipase.

5. What results show the substrate specificity of enzymes?

The observations from test tubes 1, 3, and 6 would be serving as indicatives of substrate specificity of enzymes, because the conditions in these three tubes are ideal for the digestion of their respective biomolecule—protein (hard-boiled egg white), carbohydrates (starch), and lipids (vegetable oil), respectively.

6. In tabulated form, trace the complete digestion of a ham and liver sandwich with mayonnaise, a slice of tomato, and a lettuce leaf.

Site | Secretion | Enzyme | Substrate | End product | Optimum conditions | Mouth | Saliva | Salivary amylase | Starches in the bread | Maltose | 6.7-7 pH | Stomach | Gastric Juice | Pepsin | Proteins in the ham and liver | Peptides | Very acidic pH | Small Intestine | Pancreatin (from pancreas) | Protease | Peptides obtained from fragmenting the proteins in ham and liver | Smaller peptides | Moderately alkaline pH | | | Lipase | Fats in mayonnaise, ham, and liver | Fatty acids and glycerol | Moderately alkaline pH | | | Amylase | Other starches from the sandwich, dextrins, and glycogen | Maltose | Moderately alkaline pH | | Intestinal juice | Maltase | Maltose | Glucose | Moderately alkaline pH | | | Sucrase | Sucrose | Glucose and fructose | Moderately alkaline pH | | | Lactase | Lactose | Glucose and galactose | Moderately alkaline pH | | | Peptidase | Terminal amino acids at amino end of peptides, dipeptides | Amino acids | Moderately alkaline pH |

On the Activation of Pancreatic Lipase by Calcium Chloride at Varying pH
FRITZ SCHØNHEYDER,
KIRSTEN VOLQVARTZ

Article first published online: 8 DEC 2008
DOI: 10.1111/j.1748-1716.1945.tb00291.x

Summary.
The activating influence of calcium chloride on the hydrolysis by pancreatic lipase of several saturated triglycerides and triolein has been investigated at different pH.
1. With increasing number of carbon atoms in the fatty acids of the triglycerides the optimum pH for pancreatic lipase is displaced from 7 to 8.8.
2. Addition of CaCl2 does not alter the optimum pH for the enzymatic activity towards a triglyceride.
3. CaCl2 activates the hydrolysis of all triglycerides investigated in homogeneous as well as in heterogeneous systems and both in alkaline and acid medium.
4. In the presence of CaCl2 the pH-activity range for the higher saturated triglycerides is widened one pH unit on the acid side.
5. The higher free fatty acids are supposed to depress the hydrolysis by pancreatic lipase in a high degree, and the pronounced activation by CaCl2 in experiments with higher triglycerides is explained by the removal of free acids as insoluble calcium soaps.
6. The experiments do not support WILLSTÄTTER 'S theory of the activation of lipase by formation of complex adsorbates.

Campbell and Farrell
The Behavior of Proteins: C H A P T E R7
Enzymes, Mechanisms, and Control
Chapter Outline
7.1 The Behavior of Allosteric Enzymes
How are allosteric enzymes controlled?
7.2 The Concerted and Sequential Models for
Allosteric Enzymes
What is the concerted model for allosteric behavior?
What is the sequential model for allosteric behavior?
7.3 Control of Enzyme Activity by Phosphorylation
Does phosphorylation always increase enzyme activity? 7.4 Zymogens
7.5 The Nature of the Active Site
How do we determine the essential amino acid residues? How does the architecture of the active site affect catalysis? How do the critical amino acids catalyze the chymotrypsin reaction?
7.6 Chemical Reactions Involved in Enzyme
Mechanisms
What are the most common types of reactions?
7.7 The Active Site and Transition States
How do we determine the nature of the transition state? 7.8 Coenzymes
7.1 The Behavior of Allosteric Enzymes
The behavior of many well-known enzymes can be described quite adequately by the Michaelis–Menten model, but allosteric enzymes behave very differently.
In the last chapter, we saw similarities between the reaction kinetics of an enzyme such as chymotrypsin, which does not display allosteric behavior, and the binding of oxygen by myoglobin, which is also an example of nonallosteric behavior. The analogy extends to show the similarity in the kinetic behavior of an allosteric enzyme such as aspartate transcarbamoylase (ATCase) and the binding of oxygen by hemoglobin. Both ATCase and hemoglobin are allosteric proteins; the behaviors of both exhibit cooperative effects caused by subtle changes in quaternary structure. (Recall that quaternary structure is the arrangement in space that results from the interaction of subunits through noncovalent forces, and that positive cooperativity refers to the fact that the binding of low levels of substrate facilitates the action of the protein at higher levels of substrate, whether the action is catalytic or some other kind of binding.) In addition to displaying cooperative kinetics, allosteric enzymes have a different response to the presence of inhibitors from that of nonallosteric enzymes.
How are allosteric enzymes controlled?
ATCase catalyzes the fi rst step in a series of reactions in which the end product is cytidine triphosphate (CTP), a nucleoside triphosphate needed to make
RNA and DNA (Chapter 9). The pathways that produce nucleotides are energetically costly and involve many steps. The reaction catalyzed by aspartate transcarbamoylase is a good example of how such a pathway is controlled to avoid overproduction of such compounds. For DNA and RNA synthesis, the levels of several nucleotide triphosphates are controlled. CTP is an inhibitor of
ATCase, the enzyme that catalyzes the fi rst reaction in the pathway. This behavior is an example of feedback inhibition (also called end-product inhibition), in which the end product of the sequence of reactions inhibits the fi rst reaction in the series (Figure 7.1). Feedback inhibition is an effi cient control mechanism because the entire series of reactions can be shut down when an excess of the

References: Campbell, M. K., & Farrell, S. O. (2009). Biochemistry . California: Thomson Brooks/Cole. Chaffee, E. E., & Greisheimer, E. M. (1975). Basic Physiology and Anatomy. Quezon City: MW Publishing, Inc. Garrett, R. H., & Grisham, C. M. (2010). Biochemistry. Massachusetts: Brooks/Cole. Indiana University. (2010). Retrieved March 8, 2012, from Indiana University: http://www.indiana.edu/~nimsmsf/P215/p215notes/LabManual/Lab12.pdf Lehninger, A Schonheyder, F., & Volqvartz, K. (2008). On the Activation of Pancreatic Lipase by Calcium Chloride at Varying pH. Acta Physiologica Scandinavica , 62-69. Tortora, G. J., & Anagnostakos, N. P. (1990). Principles of Anatomy and Physiology. New York: Harper & Row, Publishers.