The Effect of Pancreatic Amylase Concentration and pH on Carbohydrate Digestion and Glucose Metabolism

The Effect of Pancreatic Amylase Concentration and pH on Carbohydrate Digestion and Glucose Metabolism ABSTRACT This experiment was conducted to observe the rate of carbohydrate digestion by altering the concentration of pancreatic amylase and the pH levels in various assays. It also served to demonstrate the glucose metabolism trends in individuals with normal glucose tolerance levels as well as a subject with an abnormal tolerance level. In this experiment, starch digestion was the most efficient in the assay with the largest concentration of pancreatic amylase— 0.4444 mg/mL, whereas it took the longest in the one with the lowest concentration— 0.0556 mg/mL. Next, the optimum pH for the enzymatic activity was determined to be 7— at this pH level, the starch was digested the fastest. Lastly, after the oral glucose tolerance test was completed, it was noted that in individuals with normal glucose tolerance levels, the plasma glucose level increases for the first hour upon consumption of the sweetened drink, however, steadily decreases back to the initial fasting level. In other words, the plasma glucose levels of these subjects increased from an initial mean level of 4.9 ± 0.45mmol/L, to 8.1 ± 0.99 mmol/L at 60 minutes, but then decreased to 5.5 ± 0.28 mmol/L at the final reading. In the subject with the abnormal glucose tolerance level, the intial value was already high—7.6 mmol/L and it continuously rose and stayed around 14 mmol/L after consumption of the sweet drink. INTRODUCTION Carbohydrate digestion serves to break down consumed carbohydrate molecules into monosaccharides that are utilized by cells in the body to generate energy. Carbohydrates are comprised of starch, a polysaccharide; hence it provides a great source of energy for animals. The digestion of starch begins in the mouth of many animals as it is in the presence of an enzyme known as amylase. Enzymes are biological catalysts that are responsible for numerous chemical reactions in the body (Nichols and Cholewiak, 1991). This salivary amylase, found in saliva begins digesting the starch by breaking it down into disaccharide molecules called maltose and glucose. Later, pancreatic amylase, secreted by the pancreas, continues the digestion of these disaccharides into monosaccharide molecules to be used by the body’s cells. This experiment was undertaken to observe the effect of varying concentrations of amylase as well as different pH levels on starch digestion. Enzymes have optimum pH levels where they are the most efficient at catalyzing their reactions. At high pH levels, enzymes’ side chains tend to gain H+ ions whereas at lower levels, they lose H+ ions (Nichols and Cholewiak, 1991). This inactivates the enzymes or denatures them. During this experiment, it was predicted that larger concentrations of the pancreatic amylase would lead to faster digestion and at extreme pH levels— very acidic or highly alkaline— the amylase enzyme would denature, leading to a longer digestion process. In an experiment conducted by Mitsura et al., (1989), the digestion of raw starch by αamylase and glucoamylase was observed in a strain of mold, Chalara paradoxa. Alpha-amylase hydrolyzes or begins digesting the large alpha-linked starch molecule by splitting the central α-l4-D- glucoside linkages (Guy and Jenness, 1958). Glucoamylase also hydrolyzes glucoside linkages, and together they yield the disaccharide molecules maltose and glucose. Mitsura et al., (1989) conducted the experiment to observe the rate of starch digestion by each of these amylases on their own and combined. They found that on their own, the enzymes both digested the same starches with equal efficiency and when combined, the process was sped up. In another experiment by Yasunori et al., (1979), the effect of pH on β-amylase was observed. β-amylase initiates starch digestion in plants— it is not found in animals. Upon completion of the experiment, the optimum pH for this amylase enzyme was found to be 5.4. This value is slightly acidic; however it still supports the hypothesis that fairly neutral pH levels would be favorable as they would prevent denaturing. Conducting this experiment also allowed for the observance of glucose metabolism in subjects with normal glucose tolerance levels as well as an individual with an abnormal tolerance level. Glucagon and insulin are both produced by the pancreas. Glucagon increases the blood sugar level whereas insulin influences the uptake of glucose to be used by cells and promotes its conversion to glycogen. If the pancreas produces these hormones abnormally, it leads to low plasma glucose levels, causing hypoglycemia or high levels, leading to diabetes. The oral glucose tolerance test (OGTT) is a standardized test used to test for diabetes, and is usually given after a night of fasting (O'Shaughnessey, 2012). This test was conducted on subjects and it was hypothesized that the individual with the abnormal glucose tolerance level would maintain a high glucose level after being given the sweet drink. RESULTS FIGURES 600 Mean (± standard deviation)Time to Starch Digestion (sec) 500 400 300 200 100 0 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5 Concentration of Amylase (mg/mL) Figure 1. The mean value for the effect of various pancreatic amylase concentrations (mg/mL) on the time to starch digestion (sec). Here, n= 11 and the standard deviation varies at each concentration. At 0.05 mg/mL, the mean was 394.73 ± 114.62 sec, 189.09 ± 58.22 sec at 0.11 mg/mL, 90.64 ± 34.24 sec at 0.22 mg/mL, and 44.91 ± 20.52 sec at 0.44 mg/mL. 1400 1200 1000 800 600 400 200 0 4.5 5 5.5 6 6.5 pH 7 7.5 8 8.5 Figure 2. The mean value for the effect of pH on time to starch digestion (sec) by pancreatic amylase. Here, n= 11 and the standard deviation varies for each recorded time. At pH 5, the Mean (± standard deviation) Time to Starch Digestion (sec) mean was 1084.55 ± 353.93 sec, 84 ± 28.5 sec at pH 6, 50.45 ± 15.88 sec at pH 7 and 68.64 ± 22.15 sec at pH 8. 16 Mean (± standard deviation) Plasma Glucose Levels (mmol/L) 14 12 10 8 6 4 2 0 0 50 100 150 Subjects with Normal Plasma Glucose Metabolism Subject with Abnormal Plasma Glucose Metabolism Time (min) Figure 3. Oral Glucose Tolerance Test comparing the plasma glucose levels (mmol/L) over time (min) for a group of subjects with normal glucose tolerance levels and one subject with abnormal glucose tolerance. In the group, n= 10, and the standard deviation varies at each recorded time. At 0 min, the mean was 4.9 ± 0.45 mmol/L, 7.7 ± 0.94 mmol/L at 30 min, 8.1 ± 0.99 mmol/L at 60 min, 7.1 ± 0.73 mmol/L at 90 min, and 5.5 ± 0.28 mmol/L at 120 min. RESULTS TEXT In Figure 1, the efficiency of digestion due to varying concentrations of pancreatic amylase can be observed. The sample size was 11, therefore the mean values for the time to starch digestion were calculated. At the greatest concentration, 0.4444 mg/mL of the pancreatic amylase, digestion was the fastest—it came to a stop at 44.91 ± 20.52 seconds. The time for starch digestion to complete increased as the concentration decreased. At the lowest concentration, 0.05556 mg/mL, the digestion took the longest—394.73 ± 114.62 seconds. In Figure 2, the average values for the time to starch digestion at different pH levels were calculated. The optimum pH level was found to be 7 since digestion occurred the fastest here— 50.45 ± 15.88 seconds. At low pH levels, the digestion occurred very slowly—at pH 5 it took the longest: 1084.55 ± 353.93 seconds, however around pH levels 6 to 8, the time to digestion occurred in closer range. In Figure 3, the results of the oral glucose tolerance test can be seen. In the group with normal glucose metabolism of size 10, the average values were calculated for the plasma glucose levels. The normal subjects showed low initial levels of glucose compared to the abnormal student— 4.9 ± 0.45 mmol/L compared with 7.6 mmol/L respectively. The normal subjects all returned to a level close to their initial measure, 5.5 ± 0.28 mmol/L at the end of the experiment, however, the abnormal subject stayed around 14 mmol/L. DISCUSSION Before completing the experiments, it was predicted that higher concentrations of the pancreatic amylase would lead to faster digestion. According to the obtained results depicted in Figure 1, this hypothesis can be accepted. As stated by Worthington (1999), enzyme concentration influences the reaction rate when the substrate is in excess. Changes in the amount of product would depend solely on the amount of the enzyme present (Worthington 1999). In this experiment, the amount of the substrate was kept constant and the levels of pancreatic amylase were altered, leading to differences in the time taken for digestion to be completed. Pertaining to the influence of pH levels on starch digestion, it was hypothesized that digestion would occur the most efficiently around the neutral level, 7. This hypothesis can also be accepted as 7 was indeed the most favourable pH value, or, in other words, the optimum pH for starch digestion by pancreatic amylase. This is illustrated in Figure 2— the quickest mean time to starch digestion occurred at pH level 7. This value was also observed to be the optimum pH in many literature sources. Worthington (1999) stated that the optimum pH values vary from one enzyme to another, however, pancreatic amylase functions most efficiently at pH 6.7 to 7. In a similar experiment to this one, the optimum pH for amylase was said to be 7 (Thuvasethakul, 1974). This is due to the specific shape of the enzyme molecules. At lower pH values, there would be more hydronium ions that would attach to the enzyme’s side chains, altering its shape and hindering it useless. The opposite would happen at high pH levels— hydronium ions would break apart from the enzyme, prohibiting the substrate from binding and preventing catalysis (Nishiura, 1999). Lastly, in the experiment conducted to show the comparison of plasma glucose levels in individuals with normal glucose metabolism to a person with abnormal glucose metabolism, it was hypothesized that the normal subjects would maintain a particular plasma glucose level. In other words, in individuals who are capable of producing adequate amounts of glucagon and insulin in response to consuming carbohydrates, their plasma glucose levels should return to normal after a meal. This hypothesis can also be accepted, as the findings portrayed in Figure 3 is in support. The subject with the abnormal glucose metabolism does not return to the initial, yet high plasma glucose level. This is due to inadequate hormone production by the pancreas that is necessary for maintaining the normal levels of sugar in the body. Although the findings in these experiments were supported by various literature sources, there still could have been sources of error. An instance of error could arise from not monitoring the temperature. Chemical reactions tend to speed up as temperature increases, therefore the reactions could have been influenced by such changes. Incorrect measurements could have also been contributed to the group data—many of the deviation values were large. In the future, this experiment could be improved if the group sizes were smaller— there would be less deviation to account for, reducing some sources of error. References Bartoli, E, Fra, GP, Schianca, C (2011) The oral glucose tolerance test (OGTT) revisited. European Journal of Internal Medicine. 22: 8-12. Guy, EJ, Jenness R (1958) Separation, concentration, and properties of alpha-amylase from cows’ milk. Journal of Dairy Science. 40: 13-27. Mitsura, M, Yoshihiro, Y, Norio, K, Keiji, K (1989) Raw starch digestion by α-Amylase and Glucoamylase from Chalara paradoxa. Starch ‐ Stärke. 41: 382-385. Nichols, BD, and Cholewiak LB (1991) A quantitative enzyme study using simple equipment. ABLE. 12: 218. Nishiura, J (1999) pH and Enzymes. Brooklyn College: City University of New York. 8. O'Shaughnessey, C (2012) Hypoglycemia. Primary care reports. Web. Russel, JR, Young, AW, Jorgenson NA (1981) Effect dietary corn starch intake on pancreatic amylase and intestinal maltase and pH in cattle. Journal of Animal Science. 5: 1177-1182. Thuvasethakul, P (1974). Human pancreatic alpha-amylase. ProQuest Dissertations and Theses p. 80. Tufvesson, F, Skrabanja, V, Björck, I, Elmståhl, HL, Eliasson AC (2001) Digestibility of starch systems containing amylose-glycerol monopalmitin complexes. Academic Press. 34: 131-139. Worthington, V (1999) Effects of pH (Introduction to Enzymes). Enzymes, Biochemicals: Worthington Biochemical Corporation. Yasunori, N, Toshiko, K, Takehiko W (1979) Kinetic study of soybean β-amy1ase: The effect of pH. Journal of Biochemistry. 89: 41-45. APPENDIX In order to find the concentration of amylase in Tube D, we would have to find the concentration in the previous tubes using the formula C1V1=C2V2. Tube A: The initial concentration, C1= 2 mg/mL The initial volume, V1= 3 mL The final volume, V2= 6 mL Now we can solve for the final concentration: C2= C1V1/V2 = (2 mg/mL)(3 mL)/(6 mL) = 1 mg/mL Using this obtained concentration for the initial concentration in Tube B: C1= 1 mg/mL V1= 3 mL V2= 6 mL C2= C1V1/V2 = (1 mg/mL)(3 mL)/(6 mL) = 0.5 mg/mL Using this obtained concentration for the initial concentration in Tube C: C1= 0.5 mg/mL V1= 3 mL V2= 6 mL C2= C1V1/V2 = (0.5 mg/mL)(3 mL)/(6 mL) = 0.25 mg/mL Finally, using this concentration for the initial concentration in Tube D: C1= 0.25 mg/mL V1= 3 mL V2= 6 mL C2= C1V1/V2 = (0.25 mg/mL)(3 mL)/(6 mL) = 0.125 mg/mL