Effect of Inhibitor on Pancreatic Lipase Activity
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Published: Mon, 14 May 2018
Lipase is an enzyme that the body uses to break down fats in food into fatty acids and glycerol so that they can be absorbed in the micro villi in small intestine into the bloodstream. Lipase is primarily produced in the pancreas but is also in the mouth and stomach. Most people produce enough pancreatic lipase, but people with cystic fibrosis, Crohn’s disease, and celiac disease may not have enough lipase to get the nutrition they need from food.  Similar like other enzymes, lipases help to regulate biochemical reactions in human body. Many of those reactions might happen without the enzyme but at very slow rate. However, if an enzyme is present reaction is faster and more efficient.  Role of lipase is represented on figure 1 bellow  :
“One of the many enzymes pancreatic juice contains is lipase. As a result of the alkalinity of the bile salts, the pH of the duodenum is approximately 7.0, which is also the optimum pH for pancreatic lipase. Having been fully digested in the duodenum, the lipid-soluble fatty acids and glycerol diffuse through the phospholipid bilayer of the plasma membranes making up the epithelial cells of the small intestine. Milk is a white liquid composed mostly of water (87.3%), with small amounts of fats (3.9%), and non-fat solids such as proteins and lactose (8.8). Milk contains more fat than most liquids and a majority of these lipids are classed as triglycerides which therefore makes milk a suitable liquid to be used for this experiment. Using solid fat such as lard would be impractical because the enzyme lipase would only be able to bind with lipids on the surface of the lard, meaning there would be an extremely slow reaction rate. The globules of fat found in the milk gives the lipids a larger surface area and provides more ‘surfaces’ that the lipase enzyme can bind to.” 
Three kinds of tea, oolong, green and black, have been widely used for their purported health properties from ancient times all over the world, especially to prevent obesity and lipid metabolism.  In vitro studies with green tea extracts containing 25% of catechins have shown its capacity (in conditions similar to physiological ones) to significantly inhibit the gastric lipase, and in a lower extent also the pancreatic lipase. Thus, the lipolysis of long-chain triglycerides is reduced in a 37%. 
In this experiment, we will investigate the effect of green tea extract (inhibitor) on the pancreatic lipase activity in milk (Alpsko mleko with 3.3% of fat). Therefore our research question will be:
How do different amounts (0.5mL, 1mL, 2.5mL, 5mL, 10mL) of green tea (Camellia sinensis) extracts which can act as lipase inhibitor, affect the rate of reaction catalysed by pancreatic lipase?
As can be seen from the research question, our independent variable will be the amount of green tea extract added to a reaction and the depended variable will be the rate reaction, which will be expressed as change in pH per unit of time.
VOLUME OF MILK: milk serves as source of substrate that is needed for lipase action. In order to ensure the same amount of substrate (lipids) will be available at different amount of inhibitor, 5mL of milk will be added to the test tube at every trial.
% OF FAT IN MILK: as fats in milk are essential for examined reaction, we will use the milk with the same percent of containing fat for all measurements.
ROOM TEMPERATURE: as also temperature can affect the rate of reaction, all the measurements will be done in the same classroom, away from any heat source to ensure equal conditions for all trials. Record the room temperature several times, during the experiment to make sure, that temperature is not varying.
LIPASE CONCENTRATION: 5% solution of lipase will be prepared and used at all measurements into order to ensure the same enzyme concentration at all trials
APPARATUS USED: because different laboratory equipment have different uncertainties, make sure that you will use the same balance/pipette/micropipette etc. for the measurement of the same reagent.
lipase powder from porcine pancreas 
milk (Alpsko mleko with 3.5% of fat) 
electronic balance (± 0.001g)
computer with LoggerPro software
electronic thermometer (±0.5°C)
pipette (5mL ± 0.006mL)
pipette (10mL ± 0.006mL)
micropipette (1000Î¼L ± 0.0006 Î¼L)
measuring cylinder (200mL ± 0.5mL)
250mL beaker with stopper
volumetric flask (100mL± 0.1mL)
25 test tubes (20mL)
test tube stand
stopwatch on computer (±0.5)
3 PRELIMINARY PROCEDURES
Preparation of green tea extract
In order to prepare tea extract, measure 200mL of deionised water using a measuring cylinder and boil it. Then steep 5 tea bags into boiled water in order to obtain concentrated green tea extract. After 8 minutes take the tea bags out and pour the extract into the 250mL beaker. Seal the beaker and let the extract to cool down.
Preparation of 5% porcine pancreas lipase solution
Put the 100mL volumetric flask on the balance and reset it. Then, measure exactly 5.00 grams of lipase from porcine lipase into the volumetric flask. After that, add deionised water to the conical flask up to the margin for 100mL. Seal the volumetric flask and swirl it 5 times.
Connect LoggerPro interphase and pH probe to computer. Launch LoggerPro software.
Go to “data collection icon” and set the length of measurement to 3 minutes at sampling rate 10 measurements/minute.
Measure 5.00mL of milk into the test tube with a pipette.
Add three drops of phenolphthalein indicator to the reagents mixture. Solution will become of purple/pink colour. Stir the test tube.
Place the test tube into the stand and put the pH probe into it.
With appropriate pipette/micropipette, add 0mL/ 0.5mL/ 1mL/ 2.5mL/ 5mL or10mL of 5% lipase solution into the test tube containing milk and Na2CO3 solution and press “Start measurement” immediately after adding the lipase.
After you will start the measurement observe what will happen with the colour of the solution. During pH measurement, the time will be monitored constantly on your computer screen. Record the time at which pink colour will disappear into table 1.
Make 5 repeats for each amount of lipase added on the same graph.
Repeat the procedure for all amounts of green tea added.
DEVELOPING A METHOD FOR DATA COLLECTION
In LoggerPro, you will obtain 6 graphs with 5 curves representing the change of pH per unit of time. Make linear regression for group of curves at each amount of green tea extract added.
Write down the equations of the linear regression lines and plot only that lines into the separate graph. Comment on any apparent trends that you notice. Explain the relationship between the amounts of the inhibitor (green tea extract) added on the pH. Deduce under which conditions, the rate of examined reaction (ˆ†pH/time) was the fastest and where the slowest. Justify your answer with the explanation from the literature.
If the indicator phenolphthalein changed from pink/purple colour to colourless, what type of biochemical reaction took its place?
Calculate the average times needed for purple colour of the solution to disappear. Plot the graph: volume of the inhibitor added versus the average time needed for purple colour of the solution to disappear and deduce how presence of green tea extract as an inhibitor affect the rate of lipase catalysed reaction
Evaluate the method and suggest possible improvements.
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