Effect of temperature on pancreatic lipase on lipid digestion measured using pH sensor

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Effect of temperature on pancreatic lipase on lipid digestion measured using pH sensor. Please give proper reference to my IB student, Gina if you use this material.

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Effect of temperature on pancreatic lipase on lipid digestion measured using pH sensor

  1. 1. IB Biology HL Name: Yoojin Lee Candidate Number: 002213-067Candidate Name :Yoojin LeeCandidate Number :002213-067Date of Practical :November 18, 2010 Internal Assessment – Effect on changing the temperature of pancreatic lipase on lipid digestionResearch QuestionHow will changing the temperature of pancreatic lipase together with bile solution affect therate of digestion of milk into glycerol and fatty acids, measured using pH sensor?IntroductionPancreatic lipase1 is an enzyme that digests dietary lipids into glycerol and three fatty acidsin alkaline condition. In this investigation, milk that contains fats is used. The dietary lipid inmilk is insoluble while lipase is soluble in water. Thus, by nature, lipase cannot directly breakdown the dietary lipid, because they will form two layers. Hence, an emulsifier called the bilesalts is essential. Bile salts are amphipathic, having both hydrophilic and hydrophobiccharacteristics.2 By making the lipids soluble in water, bile salts enable lipase to successfullydigest. Figure 1 shows the chemical process of lipase activity on a triglyceride31 “Pancreatic Lipase,” Wikipedia, the freeencyclopedia, http://en.wikipedia.org/wiki/Pancreatic_lipase (accessed January 8, 2011).2 R. Bowen, “Absorption of Lipids,”Colostate,http://arbl.cvmbs.colostate.edu/hbooks/pathphys/digestion/smallgut/absorb_lipids.html(accessed January 8,2011).3 “Fatty Acid Metabolism,” 1
  2. 2. IB Biology HL Name: Yoojin Lee Candidate Number: 002213-067The dietary lipid itself is neutral in terms of acidity. However, as shown in Figure 1, when thelipase breaks down the fats, producing glycerol and three fatty acids, the pH will decrease.Thus, the pH sensor is used to measure the change in pH over time. Hence, the change in pHover time represents the rate of digestion of milk.All enzymes have optimal pH and temperature ranges. The optimal pH of the pancreaticlipase activity is around 8.4 However, milk is slightly acidic, because it is a fermentedproduct of lactic acid. Thus, in order to make the condition suitable for lipase activity, sodiumbicarbonate, a weak base, is added to increase the pH. In this investigation, the pH of the milkwill be fixed and the temperature will be altered to test how different temperatures affect therate of enzyme activity. In extreme temperatures, the enzyme might denature, so onlyreasonable temperatures ranging from 5℃ to 55℃ are tested.Natuurlijkerwijs,http://www.natuurlijkerwijs.com/english/Fatty_acid_metabolism.htm (accessed January 8, 2011).4 “Effects of pH (introduction to Enzymes),” Worthington Biochemical Corporation,http://www.worthington-biochem.com/introbiochem/effectsph.html (accessed January 8, 2011). 2
  3. 3. IB Biology HL Name: Yoojin Lee Candidate Number: 002213-067HypothesisThe rate of lipase activity is represented by the change in pH over time. Since pancreaticlipase can be found in human body, surely it works at temperatures around body temperature,36.5℃ and the optimal temperature should be close to the body temperature as well. Hence,the pH will drop constantly until the substrate, dietary lipid in milk, is completely used. Inhigh temperatures, the enzyme may denature and not function at all, thereby not changing thepH. Likewise, in low temperatures, the enzyme may be inactive, if not denatured, and thusthe pH will not drop. Hence, the optimal temperature will produce the highest rate of enzymeactivity and as the temperatures deviate from the optimal temperature, the rate will decreaseand eventually reach 0. ㅣ ㅣFigure 2 shows the predicted relationship between the rate of lipase activity and temperature 3
  4. 4. IB Biology HL Name: Yoojin Lee Candidate Number: 002213-067Variables Variables Description Method of Measuring Independent Temperature (℃) of the The lipase-bile solution were left in lipase-bile solution different temperatures at 5℃, 25℃, 35℃, 45℃, and 55℃ using refrigerator, room temperature, and water baths. The solutions were incubated for 30 minutes and were tested immediately after incubation to limit changes in temperature. Dependant Rate of lipase activity Rate of lipase activity is represented by the change in pH over time. Since the ㅣ ㅣ lipase activity produces fatty acids, it is directly proportional to the acid formation. pH was measured using the pH sensor. Also, to limit errors, the same pH sensor was used throughout the experiment. Controlled Recording initial rate As soon as the lipase-bile solution was released into the test tube, it was capped with pH sensor to record the data immediately. Amount of lipase-bile The amount of lipase-bile mixture was solution set to 5cm3. Micropipette was used to accurately measure and transfer the solution. Temperature of the All experiments were conducted at room surrounding temperature, approximately 25℃. Since the independent variable is the temperature of the lipase-bile solution, it was vital to have the same temperature of the surrounding. Volume of milk For all trials, 5cm3 of milk was tested. Micropipette was used to accurately measure and transfer the solution. Size and type of test tubes The size and type of test tubes were constant, because they can alter the surface area of milk, which is vital for initial rate. The same size and type of test tubes were used throughout. Milk Different brands of milk contain different amount of dietary lipids. Hence, milk from the same package was used throughout the experiment. Table 1 shows the independent, dependent, and controlled variables and the methods of measuring 4
  5. 5. IB Biology HL Name: Yoojin Lee Candidate Number: 002213-067Apparatus Materials  pH sensor  Milk  Micropipette (± 0.006cm3)  Lipase  Test tubes  Bile solution  Water baths  NaHCO3 solution  Refrigerator  Temperature probe  Magnetic StirrerProcedure 1. 50cm3 of 2% lipase was prepared and mixed with 20cm3 of bile solution. 2. 5cm3 of the lipase-bile prepared in Step 1 was transferred to a separate test tube and mixed with 1cm3 of NaHCO3. 3. Step 2 was repeated 14 times to produce 15 of identical lipase-bile solution samples. 4. Three lipase-bile solutions were incubated at different temperatures for 30 minutes for triplicate trials. Temperature, ℃ Preparation Method 5 Incubated in a refrigerator 25 Incubated at room temperature 35 45 Each incubated in water bath 55 Table 2 shows the preparation methods for various temperatures 5. 5cm3 of milk was transferred to a separate test tube and an incubated lipase-bile solution was added. 6. Immediately after, data was recorded using the pH sensor and Logger Pro. (While collecting data, magnetic stirrer was used to mix the lipase-bile solution and milk thoroughly for complete reaction.) 7. Steps 5 and 6 were repeated to obtain valid triplicate data for each temperature. 5
  6. 6. IB Biology HL Name: Yoojin Lee Candidate Number: 002213-067Data Collection – Quantitative Data Graph 1 shows the raw data for the effect of changing temperature on the rate of lipase activity 6
  7. 7. IB Biology HL Name: Yoojin Lee Candidate Number: 002213-067 pH(±0.05)Time, 5 25 35 45 55 t/s Trial 1 Trial 2 Trial 3 Trial 1 Trial 2 Trial 3 Trial 1 Trial 2 Trial 3 Trial 1 Trial 2 Trial 3 Trial 1 Trial 2 Trial 3 0.00 7.63 7.58 7.60 7.80 8.01 7.80 7.68 7.64 7.62 7.75 7.68 7.68 7.70 7.66 7.66 50.0 7.70 7.59 7.52 7.81 7.98 7.81 7.68 7.63 7.63 7.67 7.65 7.67 7.64 7.41 7.67 100 7.65 7.58 7.58 7.81 7.97 7.81 7.67 7.61 7.76 7.67 7.61 7.62 7.66 7.57 7.61 150 7.65 7.57 7.57 7.80 7.96 7.80 7.65 7.58 7.58 7.66 7.59 7.61 7.66 7.58 7.62 200 7.63 7.56 7.58 7.79 7.94 7.78 7.62 7.57 7.58 7.65 7.58 7.59 7.64 7.59 7.63 250 7.62 7.55 7.55 7.77 7.93 7.76 7.60 7.55 7.56 7.64 7.56 7.58 7.66 7.57 7.62 300 7.61 7.53 7.53 7.76 7.91 7.75 7.58 7.53 7.52 7.62 7.56 7.56 7.65 7.57 7.63 350 7.60 7.52 7.51 7.74 7.89 7.73 7.56 7.51 7.51 7.60 7.55 7.57 7.65 7.57 7.62 400 7.58 7.51 7.52 7.73 7.88 7.72 7.54 7.49 7.50 7.59 7.54 7.56 7.65 7.57 7.63 450 7.57 7.50 7.50 7.72 7.85 7.71 7.54 7.48 7.48 7.59 7.53 7.55 7.65 7.59 7.61 500 7.56 7.49 7.50 7.70 7.85 7.69 7.52 7.46 7.46 7.58 7.51 7.53 7.66 7.58 7.61 550 7.55 7.48 7.47 7.69 7.83 7.68 7.50 7.45 7.45 7.57 7.52 7.52 7.66 7.57 7.61 600 7.55 7.46 7.47 7.68 7.82 7.67 7.49 7.43 7.44 7.56 7.51 7.50 7.65 7.58 7.63 650 7.53 7.46 7.45 -(a) - - 7.48 7.43 7.40 7.56 7.50 7.49 7.65 7.58 7.62 700 7.52 7.45 7.44 - - - 7.47 7.41 7.41 7.55 7.49 7.48 7.65 7.58 7.63 750 7.51 7.44 7.43 - - - 7.44 7.40 7.41 7.54 7.48 7.48 7.66 7.59 7.62 800 7.51 7.43 7.42 - - - 7.44 7.39 7.39 7.54 7.46 7.47 7.66 7.60 7.62 Table 3 shows the raw data collected on Logger Pro(a) – represents uncollected data 7
  8. 8. IB Biology HL Name: Yoojin Lee Candidate Number: 002213-067Data Collection – Qualitative DataThere were no visible changes for all different temperatures. The solutions remained opaquebecause of milk and lipase solution and gave off bad odor. The solutions were yellowish-white because small amount of bile solution, which was brown in color, was added.Data ProcessingThe absolute value of the gradient of Graph 1 represents the change in pH over time. Thus, itrepresents the rate of lipase activity. The processed data is shown in Table 3 below. Rate of lipase activity, r/s-1Temperature, Trials Mean ± T/℃ Mean(a) (± 0.05) 1 3 3 SD(b) 5.00 25.0 35.0 45.0 55.0 Table 4 shows the rates of pressure increase for different hydrogen peroxide concentrations(a) Mean: average of triplicate trials for each set.(b) SD: standard deviation for triplicate trials. 8
  9. 9. IB Biology HL Name: Yoojin Lee Candidate Number: 002213-067Sample Calculations ㅣ ㅣ Calculation of the mean rate of 5℃ lipase-bile solution from the triplicate trials. Mean ( ) = = s-1 Calculation of the standard deviation of 5℃ lipase-bile solution from the triplicate trials Standard deviation == = s-1 9
  10. 10. IB Biology HL Name: Yoojin Lee Candidate Number: 002213-067Data Presentation y = -3E-07x2 + 2E-05x + 0.0002Effect of Changing Temperature on the Rate of Lipase Activity R² = 0.7577 0.0006 0.0005 Rate of Lipase Activity, r/s-1 0.0004 0.0003 0.0002 (a) 0.0001 0 0 5 10 15 20 25 30 35 40 45 50 55 60 Temperature, T/℃ Graph 2 shows the processed data of average rates of evaporation against the number of the carbon chain. (a) Vertical error bar shows the standard deviation of the triplicate trials for the rate of evaporation. 10
  11. 11. IB Biology HL Name: Yoojin Lee Candidate Number: 002213-067ConclusionThe data suggests that the optimal temperature for lipase activity is close to 35℃ and that myhypothesis was valid as Graph 2 seems to be similar to Figure 2, which is my predictedoutcome. Also, the data shows that the rate of lipase activity decreases as the temperaturedeviates, both decrease and increase, from the optimum, which the parabolic trend linesuggests as well. For instance, the maximum rate occurs at 35℃ while the rates at 5℃ and 55℃are ostensibly lower than the rate at 35℃. The R2 value tells that there is a correlation.However, the limitation of this investigation is that the exact optimal temperature cannot befound out. Although 35℃ lipase-bile solution produced the highest rate of enzyme activity,the exact optimum may not be 35℃. Hence, the optimum temperature range is between 25℃and 45℃. According to online research, the exact optimal temperature is found out to be37℃, which is very close to the empirical data in this investigation.EvaluationThis experiment is justifiable because reliable triplicate trials were obtained. Yet, the dataconsists of wide uncertainties, partly because the rate itself was too small ranging from to . Since the fatty acids did not lower the acidity a lot, the changein pH was very opaque. Because the change in pH was hard to detect, it naturallyaccompanied a great uncertainty in measurement.Moreover, in terms of procedure, it accompanied greater uncertainty, because temperature isalways changing. Even though the experiment was immediately performed after incubation at 11
  12. 12. IB Biology HL Name: Yoojin Lee Candidate Number: 002213-067certain temperature, it will nevertheless assimilate into the room temperature as theexperiment proceeds. Hence, the greater uncertainty is present for the temperature, but theextent is unknown.Limitations and ImprovementsLimitations ImprovementsThe temperature could not be fixed as theexperiment proceeded. Although the lipase- In order to prevent temperature assimilation,bile solutions were incubated enough, as a more advanced method is needed. Perhaps,soon as the incubation was over, the if the whole experiment was done inside thetemperature inevitably started to assimilate incubator, it could prevent the temperatureinto the room temperature. This would have change.produced the major error, since temperatureitself is the independent variable.Only certain range of temperatures could betested due to technical limitations. For Better equipment is needed to test a varietyexample, 15℃ could not be tested, because of temperatures. If more time was given, this research could be further investigated bythe room temperature was around 25℃ and narrowing down the temperature ranges andthe water bath temperature range starts from finding the empirical optimal temperature.the room temperature. Lacking diversity in Then, the percent error could be calculated totemperature is another major limitation. Due make the investigation more justifiable andto time constraints, specific temperatures reliable.could not be tested. To reduce human errors, more advanced apparatus has to be used. To obtain accurateSince the test tube had to be manually capped data, the pH has to be measured as soon aswith the pH sensor, it inevitably included the lipase-bile solution hits the surface ofhuman error because of human reaction time. milk, because the reaction startsThus, the initial rate might not be accurate. instantaneously, even if the rate of lipase activity is low. Table 5 shows the limitations and the improvements 12
  13. 13. IB Biology HL Name: Yoojin Lee Candidate Number: 002213-067Bibliography1 “Pancreatic Lipase.” Wikipedia, the freeencyclopedia. http://en.wikipedia.org/wiki/Pancreatic_lipase(accessed January 8, 2011).2 Bowen, R. “Absorption of Lipids.”Colostate.http://arbl.cvmbs.colostate.edu/hbooks/pathphys/digestion/smallgut/absorb_lipids.html(accessed January 8, 2011).3 “Fatty Acid Metabolism.”Natuurlijkerwijs.http://www.natuurlijkerwijs.com/english/Fatty_acid_metabolism.htm (accessed January 8, 2011).4 “Effects of pH (introduction to Enzymes).” Worthington BiochemicalCorporation.http://www.worthington-biochem.com/introbiochem/effectsph.html (accessedJanuary 8, 2011). 13

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