A2 coursework- The effects of bile salts on digestion of fat

Planning:

        It is important to understand what fat it, where and how it is broken down in the human body.

        Fats which are usually known as triglycerides are a group of lipids.  From my As work I learned that triglycerides are made of three fatty acid molecules joined to one glycerol molecule by a condensation reaction and the reverse of this is a hydrolysis reaction.  Fatty acids are organic molecules which all have a –COOH group attached to a hydrocarbon tail.  Triglycerides are insoluble in water (they will not mix) because unlike water which is polar, the fatty acid tails have no uneven distribution of electrical charge and so the triglycerides can be said to be non-polar and so hydrophobic.

                                           

             Fig. 1.1

             Simple diagram of structure of a triglyceride molecule

        

Fig. 1.2

Diagram of a triglyceride molecule showing atoms and bonds involved

From my A2 work I have learnt that fat digestion and absorption can only occur with the aid of bile. Bile is produced in the liver but stored in the gallbladder, and enters the duodenum via the bile duct.  Substances such as phospholipids, water, cholesterol, bilirubin, bile acids, and electrolytes brought into the liver from the small intestine via the hepatic portal vein are used to make bile.  When bile is needed to aid fat digestion the process of Cholecystokinin (CCK) which is secreted from the mucosal epithelial cells in the duodenum acts on the gall bladder and bile is secreted into the small intestine and stimulates pancreatic lipase to be secreted.

During fat digestion it is important that the fat is emulsified into smaller particles so that the enzyme lipase can act on it.

 “Because fat does not dissolve in water, the fat molecules enter the duodenum in a congealed mass, which makes it impossible for the pancreatic lipase enzymes to attack them, since lipase is a water soluble enzyme and can only attack the surface of the fat molecules.  To overcome this problem the digestive system uses a substance called bile; Bile becomes suspended in the watery contents of the digestive tract.  Emulsification allows lipase to gain easier access to the fat molecules and thus accelerates their breakdown and digestion.”

The liver is the largest gland in the body, it outtakes an estimated 500 functions.  The liver is important in fat digestion because it produces bile by the oxidation of cholesterol (bile does not contain any digestive enzymes).  Bile which is stored in the gall bladder contains bile salts (also known as bile acids) and these emulsify fat to form small oil droplets called micelles (these have a larger surface area).  Bile salts aid fat digestion so that the pancreatic lipase enzyme can digest triglycerides to fatty acids and glycerol in the duodenum.  

 

Fig.1.3         

Diagram of the livers position in the body

The liver is made of two main lobes which are then subdivided into thousands of lobules.  In the centre of each lobule there is a branch of the hepatic vein.  Blood flows from the hepatic artery and hepatic portal vein through the lobule into the hepatic vein.  Hepatocytes (liver cells) are lined two cells thick in rows in the lobules so they are close to the blood, blood flows between the rows in the sinusoids.  “Other channels carry bile, which is produced by some of the hepatocytes; these channels are called bile canaliculi.  The bile flows from the centre of the lobule towards the outside, where it enters a branch of the bile duct”.  The sinusoids are lined with large phagocytic macrophages which destroy bacteria brought to the sinusoids from the hepatic portal vein, these are called kupffer cells.

Fig. 1.4

Diagram of a liver lobule

The liver has an enormous variety of functions as it is involved in many different metabolic processes.  The following list is a summary of the main functions of the liver:

• processing digested food from the intestine
• controlling levels of fats, amino acids and glucose in the blood 
• combating infections in the body 
• clearing the blood of particles and infections including bacteria 
• neutralizing and destroying drugs and toxins 
• manufacturing bile 
• storing iron, vitamins and other essential chemicals 
• breaking down food and turning it into energy 
• manufacturing, breaking down and regulating numerous hormones including sex hormones 
• Making enzymes and proteins which are responsible for most chemical reactions in the body, for example those involved in blood clotting and repair of damaged tissues.

Once the fat is broken down, the fatty acids are stored or oxidized for energy in the adipose tissue or muscle tissue and the glycerol is absorbed by the liver and is either converted into glucose (gluconeogensis) and/or used to help the breakdown of glucose into energy (glycolysis).  “The liver plays a key role in the homeostatic control of blood glucose, by storing or releasing it as needed, in response to the pancreatic hormones insulin and glucagon”.

Lipase is a digestive enzyme and from my work at As level I learned that enzymes are globular proteins.  They are made of many single amino acids, in the primary structure, linked together by the loss of a molecule of water to form peptide bonds, this reaction is known as a condensation reaction and then these make polypeptide chains, which make globular proteins.  “Like all globular proteins, enzymes are coiled into a precise three-dimensional shape, with hydrophilic R groups (side chains) on the outside of the molecule ensuring that they are soluble”.  So lipase is a soluble molecule, however fat is not and so this is what makes it difficult for the lipase to act on the fat but it is bile salts that aid this reaction to allow fat digestion to occur.  

     

Fig. 1.5

Shows a condensation reaction forming a peptide bond

Fig. 1.6

Shows enzyme-substrate complex

          Enzymes speed up chemical reactions by lowering the activation energy and not being used up themselves permanently.  Activation energy is the minimum amount of energy needed to make a reaction take place. An enzyme has an active site to which a substrate is complimentary to and so must fit into perfectly, temporary bonds occur between the substrate and some of the R groups of an enzyme and this is called an enzyme-substrate complex.

Fig. 1.7

Diagram to show lowered activation energy required with an enzyme present

        As it can be seen from the above diagram that when an enzyme is involved in a reaction then the activation energy is considerably lowered so less energy is required for the reaction to take place so this speeds up the reaction.  Without an enzyme the reaction will occur but extremely slowly.  So lipase is important in fat digestion however in this investigation it is not the lipase that I will be changing and it is not the effect of lipase on fat digestion that I will be investigating and so the amount and concentration of lipase must be kept constant.  There are many factors that effect enzyme activity that need to be kept constant in the experiment so that lipase does not become the independent variable of this experiment.

Prediction:

I predict that as the concentration of bile salt is increased then the pH will decrease further because with an increased amount of bile salt then more fat will be emulsified meaning there will be a bigger surface area for the lipase to act on. "Emulsification allows lipase to gain easier access to the fat molecules and thus accelerates their breakdown and digestion.”

  The pH will decrease because as the fat molecule is broken down into three fatty acids and one glycerol then the fatty acids have the ability to donate H+ protons making the solution more acidic (Bronsted-Lowry).  I predict that the rate of reaction will be quickest at the start of the reaction because there will be maximum number of enzyme-substrate complexes as there will be maximum amount of substrate for the enzyme to act on.  I also predict that the rate of reaction will be fastest for the reaction with the highest concentration of bile salt used and this is because there is more bile salt to emulsify the fat and so there will be more manageable sized substrate for the enzyme to act on.  The pH change will be the biggest decrease for the concentration with the highest concentration of bile salt used and this will be seen from my graph.  I can also then calculate the rate of reaction within a time for the reactions to prove my prediction correct if so.

Safety:

        The experiment should be kept organized and so as safe as possible.  There are a number of risks that should be addressed before out taking the experiment.  Bags, books and folders should be placed away from work areas to ensure there is less risk of trips n falls.  Work areas should be kept as organized as possible at all times so that not only will the experiment be at reduced risk but also there is reduced risk to myself and others around.  The solutions that will be handled are lipase, bile salt and water.  

        Lipase can cause irritation to skin so hands should be washed when there are spillages and work areas if there are spillages on them.  Spillages on the floor should be mopped up.

        Bile salt if ingested can make you sick and is so medical advice should be seeked immediately.  Once again any spillages should be cleaned up to prevent slips happening.  

        Water should be mopped up when spilt but also there will be electrical socket nearby to the water bath and so the water bath should be kept at distance to the sockets.  Care should be taken at all times.

        There will be a lot of glassware and if broken then these should be cleaned up using a dust pan and brush and broken glass should be disposed of into the broken glass bin.

        Care should be taken when handling the solutions so that contamination doesn’t occur as this could affect the results obtained.

        Lab coats and goggles should be worn at all times to protect clothing from the equipment and vice versa and to protect eyes.  If contact of solution with eyes does occur then eyes should be rinsed out with water and medical attention seeked if necessary.

Equipment:

        Below is a table of the equipment used in my experiment.

Diagram of experiment:

Method:

1. Put on goggles and lab coat.  Clear up all work areas.
2. Prepare a results table that repeats can be included in.
3. Collect all equipment needed as shown in equipment table
4. Prepare the water bath at 40degrees and use a thermometer to check the temperature
5. Prepare the different concentrations as shown in the below table: (do not add the cream yet!)

6. Place these 5 boiling tubes into the water bath in the boiling tube rack
7. Calibrate the pH probe in the buffer solution
8. Now add 2ml of cream to each test tube
9. Place the pH probe into the boiling tube and record pH recording immediately and record this as 0 minutes in table
10. Start stop watch
11. Record pH of every boiling tube after 2 minute intervals and record in table up till 30 minutes
12. Now repeat each concentration 3 time

Fair testing/ control variables:

        To achieve ‘true’ and reliable results form this experiment then there are variables that should be controlled.  The only variable that should change is the independent variable which is the concentration of the bile salt.  Below are all the variables that should be kept constant and controlled to make this experiment fair.

• Measuring methods:  from my preliminary work I found the best way to measure solutions for this experiment was to use measuring cylinders and pipettes.  I should not change these because different equipment shows different meniscus sizes.  I used small scale measuring cylinders (10ml) for greater precision.
• Temperature of the surroundings:  if the temperature of the surroundings is changed then this can affect the activity of the enzyme lipase.  With increased temperature the enzyme will work better but with drastic changes then the R group of the active site will be changed and so the enzyme will be denatured.  Lowered temperature can decrease the rate of enzyme activity and so decrease the rate of reaction.  However the temperature of the surroundings will be kept the same as the experiment will be completed in one day and so the weather should stay the same and no heaters will be turned on or off.
• Same pH probe:  the pH probe will be kept the same throughout the experiment because they may work slightly different as one may be older than others and this will affect ph readings so the same pH probe will be used.
• Same food type:  the same food type will be used, cream, because different food substances contain different amounts of fat and so will affect pH.
• Volume of food substance:  the same volume of food substance will be used because obviously the greater the volume of food substance then the greater the amount of fat and so this will affect the pH reading.  2ml of cream will be used for every individual test.
• Temperature of water bath:  temperature will affect the activity of the enzyme lipase.  The water bath will be kept constant at 40 degrees by checking the temperature constantly using a thermometer.  If the temperature is decreased then this will slow down the rate of the reaction and so the lipase will not be breaking down the fat as quickly and so temperature will become an independent variable in this experiment.  I found from my preliminary work that using a water bath worked well and it will keep the temperature more constant then a Bunsen burner which will also be time-consuming as I will have to keep turning the Bunsen burner on and off because air heats up and cools down quicker than water.
• Time length:  the overall time length (30 minutes) should be kept the same for each individual test because further changes in pH could occur after 30 minutes and so then the results can not be compared for the different concentrations of bile salts.  Also I found from my preliminary work that 2 minute intervals work well and these should be kept the same for each individual test because further pH changes could occur and so then different concentrations of bile salt could not be compared.
• Concentration of lipase:  the concentration of lipase should be kept the same because with an increased concentration of lipase then the rate of reaction will occur quicker and so the reaction may finish within 30 minutes and then there will be no more fat to be broken down and so the pH will not decrease any further.  So the concentration of lipase should be kept constant through out the experiment using a volume of 2ml of lipase.
• Clean equipment:  equipment should be cleaned thoroughly before adding solutions because if not cleaned then the equipment may contain solutions and so contamination may occur.  Also for example if a boiling tube is not cleaned out properly then there may be bile in it and then this will mean that the right amount of concentration and volume for that individual test will not be used.

Variables:

• Dependent variable:

 The dependent variable is dependent upon the independent variable so, hence when the concentration of the bile salt (independent variable) changes then the pH (dependent variable) will change.  It is important that all control factors are kept constant and only the concentration of the bile salt is changed so that pH is only dependent upon the concentration of bile salt and not any other factor so that I receive reliable results.  I will use a ph probe to record the pH change after every 2minute intervals to two decimal places.

• Independent variable:

The independent variable is the only variable in the experiment that needs to be changed.  The dependent variable is changes upon this.  The independent variable of this experiment is the concentrations of the bile salt which I will be changing and the pH (dependent variable) will change according to this.  So it is important that the concentrations of the bile salts are the only variable changed in the experiment to gat reliable results.  I will change the concentrations of the bile salt to the concentrations shown in the blow table. These need to be changed to investigate the aim to see if bile salts do aid the digestion of fats.  So hopefully by using these different concentrations I will achieve different pH readings which I can compare to investigate the aim.

Analysis:

From looking at both my results table and plotted graph it can be seen that my prediction has been proven right.  This is that when the concentration of bile salt is increased then there is a decrease in pH (more acidic due to increased concentration of hydrogen ions).  

        This is shown for instance when looking at the graph that when 100% concentration of bile salt is used then the end pH (30 minute reading) is 7.48pH and when 25% concentration bile salt is used the 30 minute pH reading is 9.45 so this shows that when a higher concentration of bile salt is used then the pH is lower (more acidic).

The decrease in pH achieved when fatty acids are broken down from fat molecule.  When the gall bladder releases its stored bile into the small intestine then bile emulsifies the fat into smaller more manageable sizes with an increased surface area for the enzyme lipase to act on more easily and quicker (so there’s an increased rate of reaction).  Lipase breaks down the fat molecule into 3 fatty acid molecules and one glycerol molecule.  The fatty acids from the broken triglyceride molecule make the solution more acidic.  So hence my prediction has been proved right that bile salts do aid the digestion of fats.

        From looking at my graph I have plotted to show the pH change for different concentrations of bile salt reacting with fat and lipase in 30 minutes, it can be seen that as the reaction proceeds then the rate of reaction decreases and this is evident as shown that the lines start to level out and there is not such a big decrease in pH as there was at the beginning of the reaction.  This could be due to a number of factors.

        At the beginning of the reaction there is maximum amount of substrate and maximum amount of enzyme and so there are more enzyme-substrate complexes than at any other time of the reaction.  However as time goes on there comes to point where the rate of reaction is constant due to a limiting factor.  The limiting factor in this instance is concentration of enzyme “there comes a point where every single active site is continuously working, if more substrate is added the enzyme cannot work faster.  Substrate molecules are queuing up for an active site to become vacant.  The enzyme is working at its maximum possible rate, known as Vmax”.  So there is a decreasing number of enzyme-substrate complexes so the rate of reaction slows down.

        

        The slowing down of the reaction could also be due to the change of pH.  Enzymes have an optimum pH at which the rate of reaction is fastest.  Maybe the optimum pH of lipase was between pH10-11 because all 5 of the concentrations started with a pH between this and this was when the reaction was the fastest (the start of the reaction) as there was the biggest pH drops occurring then.

        “Acidity and alkalinity can affect the active site of an enzyme.  Free hydrogen ions can affect the charges on the amino acid side-chains of the enzymes active site.  This will affect the hydrogen bonding and so change the three-dimensional shape of the enzyme and the shape of its active site.  The substrate will no longer fit the active site, the enzyme loses its activity and the rate of reaction falls”.

        So when the pH increasingly becomes acidic due to the fatty acids that have been formed from the breakdown of triglycerides then this could affect the enzyme activity and so could explain the levelling off of the lines as the enzyme could now be becoming denatured.  

        There are other factors that could have affected the activity of the enzyme activity such as temperature; however the pH of the solution is more significant than this because it was known that pH would be changing as it was pH that I tested and temperature was kept constant as possible by using a water bath set at 40degrees.

        From looking at my graph I can see some anomalous results that do not fit the trend of the best fit line.  For instance when 100% concentration of bile salt is used at 12 minutes there is an anomalous result and this could be again due to a number of factors.

To support my hypothesis further I have calculated the rate of reaction within the first 4 minutes for each of the different 5 concentrations of the bile salt used.  My calculations and results of rate of reaction are shown in the table below:

In the above table it can be seen that as concentration of bile salt is increased then the rate of reaction is increased.  At 100% concentration of bile salt there is a rate of reaction of 12.30s-1 compared to when 0% concentration of bile salt is used there is a rate of reaction of only 3.35s-1   and this is a big difference of 8.95 s-1.

I also plotted a graph to show these calculations to see if there was a correlation.  Like I had suspected it was shown that there was a positive correlation between the concentration of bile salt used and the rate of reaction within the first 4 minutes. The graph was 1st order meaning that the concentration of bile salt was proportional to the rate of reaction.

Fig. 1.8

First order

 I think this was because there was more bile salt to emulsify the fat then this created an increased surface area for the lipase to work on and so increased the rate of reaction.

        I feel I have investigated my aim well enough to now say I have proved my prediction right.

Evaluation:

        I feel that I have investigated my aim well however if I were to outtake the investigation again I would consider making some improvements so that maybe I would not get anomalous results as I did and to improve the accuracy of my results.  

        From my results I was able to prove my prediction correct so the errors of some results were not that significant, however they were still errors and so should be addressed.  They were slightly off the best fit line.  There are a number of factors that could have resulted to these errors occurring.

        One of these could be that due to the time limitations then I had to use the same pH probe to test 5 different concentrations of bile salt in 30 minutes.  The significance of this is that when after every 2 minutes I did not have time to clean it in a buffer solution and so when I was testing the pH of one concentration, the pH probe could still have the solution from the last boiling tube on its surface and this could have affected the pH readings I received, recorded and plotted.  This could explain some of the slightly higher or lower pH readings I received that stood out from the best fit line. If I was to do the experiment again I would calibrate the pH probe in a buffer solution (distilled water) as this would make the experiment fairer but also give me more reliable results that were also more accurate.  Also the lipase may have affected my results because there might have been fluctuations in the surrounding temperature however I don’t feel this is significant enough because we out took the experiment on the same day so temperatures could not have changed that significantly however a improvement could be to make sure that the lipase was prepared as close as possible to the time the experiment started so that the lipase did not experience significant fluctuations of temperature.

        There may have been an improvement to the equipment used.  If I was to out take the experiment again I would use a glass pipette instead of a measuring cylinder because when transferring the solutions maybe some solution did not get all transferred as it stayed in the measuring cylinder.  If for example there was less lipase transferred then measured then this could be significant to slow the reaction down depending upon how much enzyme was left in the measuring cylinder and so not as many enzyme-substrate complexes could form and so at that time there were not as many triglycerides broken down as there should have been and so the pH was not as acidic as it should have been.

        In the future to receive more comparable results I would include more concentrations of bile salt used, so that I could see again where and why anomalous results occurred.

        To make the experiment more reliable I would consider using more pH probes so there was one for each concentration and I think this would improve the accuracy of my results and also make it more reliable because it would prevent contamination of different concentrated solutions of bile salt.

        I would also consider using more methods of comparing my results to investigate my aim further.  Maybe I would use spearman’s rank to test for correlation.  I would use my pH results and compare them against bile salt concentration or I could compare bile salt concentration to rate of reaction to test for positive or negative correlation if any.

        I would like to have included more repeats so in the future I would allow myself more time to carry out the experiment so this could be done.  However I did use repeats in this experiment and I averaged these results out and plotted a graph using these.

        There were limitations in my experiment that were that everyone had to make up their own solutions and so everyone’s solutions may have slight changes in concentration of bile salt actually contained and so people could receive different pH results due to this.

Another limitation was that there were only 2 water baths for the class to use and so sometimes it was hard to get to the water bath after every 2 minute intervals exactly as there were other people in the way and so this could mean that the pH was not recorded at exactly 2 minute intervals.

        Another limitation in this experiment was that due to time limitation I had to measure the pH of 5 different concentrations within 30 minutes and so it was impossible to measure the time of the reaction to the nearest 0.1 second after 2 minute intervals as it was such a tight time gap to measure the pH for 5 different concentrations even though the stopwatch showed the measurements to the nearest 0.01 second.

        Even though there are possible improvements to the investigation I think that the results I obtained were significant enough to use to investigate my aim as I was able to draw up graphs and compare trends to prove my prediction correct that at when a higher concentration of bile salt is used then there is a bigger decrease in pH.  So my anomalous results were not that significant enough that I was unable to draw up conclusions.  Even though the anomalous results can be seen on my graph they are not a great distance away from the best fit line and so this shows the experiment worked well.

Appendix 1

Calculating rate of reactions:

For the first 4 minutes of the averaged pH recordings of the different concentrations I have calculated the rate of reaction as I think this will help me to investigate my aim.  Below are my results:

References:

Websites:

Books: 

• OCR book, Mammalian physiology and behavior, page 19
• OCR biology 1 book page 43

• Biology 1 book OCR, page 46

• Advanced biology for you book, Gareth Williams, page 69.

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