The Inhibition of Lipase Milkfat Digestion in the Presence of Orlistat (Tetrahydrolipstatin).

Introduction

(First published 10/10/2016)

School practical science rarely bears any resemblance to modern day experimental science and often, for the students, the principles being demonstrated are lost in the oversimplified and dull experiments they are expected to perform. This hardly develops the hands-on, practical and analytical skills universities are so desperately looking for in prospective students, or adequately stretches and challenges the more able.

In many modern chemical kinetics experiments huge numbers of measurements are taken and the whole process is conducted by robots. This removes human error from parts of the experiment particularly prone to it (for example pipetting) and speeds up the process considerably. An excellent example of a modern chemical kinetics experiment would be a high throughput screen, for enzyme inhibitors or drugs. However with machines doing huge numbers of repetitive tasks, experimental design becomes much more crucial.

Once an experiment has been designed, a robot has mixed the samples together and chemical reactions initiated, rate measurements can be taken. Many experimental systems exploit fluorescence laser spectroscopy. Here analysis is relatively free from interference from other factors and because of the popularity of the method, multi-well plate readers have become widely available and are reasonably cheap.

The multi-well plate experimental format is an innovation in itself facilitating two important trends in modern science, miniaturisation and high throughput. Typically this format will allow you to conduct 384 tiny kinetic experiments (10-20 µL) simultaneously. Obviously the potential to generate data is huge and again modernisation in the use of computers for the processing and analysis of data is vitally important.

Wouldn’t it be good if current school practical experiments involving enzymology or chemical kinetics reflected contemporary methods in the respective disciplines to some extent?

Towards these ends I have been developing an experiment which is simple, safe, cheap and very robust. In conducting it, students will gain a good taste of modern enzymology and some of the types of method currently used. I will explain the experiment in its simplest form but it should be emphasised that throughout the experimental design there are fantastic opportunities to get students to work through a range of important practical scientific issues.

The experiment itself uses ideas from the classic “disappearing cross” experiment[1] to estimate rates and can be conducted using the standard school lab dropping tiles as multi-well plates. It can also use a smart-phone clamped into place as a spectrometer which will photograph or video record the experiment, but digital cameras can be used just as effectively, or if nothing is available you could “estimate” using the human eye as your measurement device. A set of micropipettes is the only thing that might require large expenditure and for most schools not having these will be the major obstacle. However a few sets would be a worthy investment considering the ease, accuracy, good training for students and significantly enhanced safety they afford.[2]

Experimental method[3]

Required solutions:[4]

  • 10x Buffer (5 mM NaxHyPO4 [x + y = 3], pH 8.0 at 1x dilution)[5] made by mixing 5.3 mL of 100 mM monobasic sodium phosphate with 94.7 mL of 100 mM dibasic sodium phosphate and diluting the whole to 200 mL with water in a volumetric flask. This gives 200 mL of a 50 mM (10x) sodium phosphate buffer solution. You should take 1.00 mL of this 10x buffer and dilute to 10.0 mL with water to check the actual working pH at 1x (5 mM) dilution (it should be pH 8).
  • Stock 10x enzyme[6] solution in 1x buffer (0.5 mg/mL).[7]
  • Full fat milk (3-5 % butterfat).
  • Stock solution of inhibitor Orlistat[8] (£82/g, 496 g/mol, 200 µL per 2 students[9]10.0 mM in 1x buffer). Make up dilutions of this in a few Eppendorf tubes: 20 µL of stock 10.0 mM and 180 µL of 1x buffer (giving 1000 µM); 20 µL of 1000 µM and 180 µL of 1x buffer (100 µM); 20 µL of 100 µM and 180 µL 1x buffer (10 µM); 20 µL of 10 µM and 180 µL 1x buffer (1.0 µM); 20 µL of 1.0 µM and 180 µL 1x buffer (0.1 µM).

There are 12 wells on a dropping tile of the type commonly used in most school labs.[10] Into the bottom of each well using a fine, waterproof marker pen draw a letter or some other symbol.

Set up a camera-phone above your tile and clamp it into place. Turn on the camera, focus it and take a photo of your tile. Mark the exact location of your tile on the bench and do not move the camera-phone or tile until the experiment is complete.

Fill 6[11] wells with 40 µL of the 10x buffer, 360 µL of milk, and then to each of the 6, add 50 µL of one of the 6 inhibitor concentrations and label them accordingly. Into a seventh well add 40 µL of 10x buffer, 360 µL of milk, 50 µL of 1x buffer; and to an eighth add 40 µL of 10x buffer, 360 µL of milk, 100 µL of 1x buffer. Add any additional controls you feel you may need (there should be 4 remaining wells available if you use 1 x 12 well dropping tile).

As quickly and efficiently as possible start the camera recording or take your first photo and then add 50 µL of enzyme to each well except the 8th (control- no enzyme), mixing each immediately after adding the enzyme. All wells must be clearly visible in the output of the camera.

Continue recording or taking photos every 30 seconds until you can identify the symbol drawn[12] into the bottom of each well except those that have no enzyme added which should remain turbid.

Wash the dropping tile and repeat if you need to collect 2 more sets of data.

Take a sample of milk diluted with the relevant buffers and carry out a serial dilution of this until a symbol is just visible below the solution in the well. Assuming you are starting with a 4 % milkfat suspension[13] what is the percentage milkfat content where the symbol becomes visible?

Analysis and conclusions

Use a spreadsheet for your data analysis. For each well calculate the actual assay concentrations of all the components added to the mixture.[14]

For each well on your plate, it is on the video when enzyme was added and the components mixed. Record the start time for each well from the video clock (when mixing is initiated). Run the video until you can make out the symbol drawn below each well and note the time down. Subtract the start time and this should give you the overall time taken from mixing to a visible sign for each well.

For the repeated experiment calculate the estimated rate of reaction in terms of the consumption of milkfat. Calculate an average rate for the repeated experiments, i.e. average for each inhibitor concentration and for each control. Plot average rate vs inhibitor concentration including error bars on your graph.[15] Table 1 shows what a set of results should look like. In this case rates have been converted to fractional activities for the enzyme by dividing all the rates in a set by the highest rate.

Table 1, showing calculated fractional activities for each concentration of orlistat.

slide2

Table Notes: 1) the entry with no enzyme is taken as the 10 mM inhibitor concentration, the negative control; 2) to make this data plot successfully in Excel, 0.001 µM is taken as the concentration with zero added inhibitor; 3) fractional activity is calculated by assuming the milk fat content is 4 %, therefore the assay percentage of milk is 2.88 % (360 x 4 /500), from the experiment to see the turbidity of the milk when a symbol can be seen, this percentage milk was used and subtracted from the 2.88 %, we then take this modified percentage and divide by the number of seconds it took for the symbol to appear and it gives a rate in terms of percentage milkfat consumed per second, then all the values in a set of experiments are divided by the highest rate this gives a fractional activity (between 0 and 1).

If this data is plotted it should look something like Graph 1[16] (processed in Excel, a copy of this spreadsheet is available to download).[17]

slide1


Notes

[1] See http://www.rsc.org/learn-chemistry/resource/res00000448/the-effect-of-temperature-on-reaction-rate?cmpid=CMP00000518

[2] See http://www.timstar.co.uk/bt140555-adjustable-micropipettes.html

[3] a) Do not under any circumstances attempt this unless under the supervision of a trained professional chemist or biologist. It is entirely the responsibility of all people conducting this experiment or adaptations of it, to carry out their own full risk assessment; b) A video demonstration of this experiment will be made available shortly and posted here.

[4] This experiment could easily be adapted to measure reaction rates as a function of enzyme concentration or temperature. Substrate concentration is probably going to be problematic using the “appearing symbol method.” During the course of the reaction the experiment generates fatty acids which will change the pH. The method as is, tries to minimise the effect of this with buffering (5 mM Phosphate, pH 8).

[5] See http://info.gbiosciences.com/blog/bid/197554/Biological-Buffers-pH-Range-and-How-to-Prepare-Them

[6] Pig pancreatic lipase powder (£ 16/25 g) from http://www.timstar.co.uk/product-range/biology/ez81571-lipase-powder.html

[7] It may be necessary to dilute this further (with 1x buffer) to slow the reaction down to a rate that is measurable given the method being used.

[8]See http://www.sigmaaldrich.com/catalog/search?term=Orlistat&interface=Product%20Name&N=0+&mode=mode%20matchpartialmax&lang=en&region=GB&focus=productN=0%20220003048%20219853286%20219853121

[9] For a class of 20 students working in pairs, 2 mL of a 10 mM solution containing roughly 10 mg is sufficient. From this you can easily run 100 such classes for the initial outlay on the inhibitor of £82, (82 p/class).

[10] See: http://www.nuffieldfoundation.org/practical-biology/investigating-effect-ph-amylase-activity

[11] It is possible to run this experiment in triplicate, including all the controls in triplicate, by putting two 12 well plates together and doing the triplicate experiments in 1 go.

[12] The first appearance of the symbol is important since we are trying to estimate initial rates, an average over the first minute or so. Chemical reactions generally slow down as the reactants become less concentrated over time.

[13] Milk fat consists of a diverse mixture of triglycerides which varies from sample to sample and species to species. No attempt has been made here of working out concentrations of milkfat in molar terms.

[14] For example sodium phosphate buffer stock solution was 50 mM. 40 µL was added to 360 µL of milk making a total volume of 400 µL of 5 mM phosphate and to this was added 100 µL of 5 mM phosphate buffer. Therefore the assay concentration of phosphate buffer was 5 mM. It is often convenient to use an equation to calculate final concentrations. (Ci x Vi)/Vf = Cf, where Ci is the initial concentration of the thing being diluted, Vi is the volume being diluted, Vf is the volume of the thing being diluted plus the volume of what you dilute it with and Cf is the concentration of stuff you are trying to calculate. So for the buffer above (50 mM x 40 µL)/ (360 + 40 µL) = 5 mM. When a 5 mM solution is then added to more 5 mM solution of the same thing, the concentration of that thing doesn’t change.

[15] There are many variations of this experiment possible. For example using photographs of your plate, it would be possible to load these into ImageJ (freely available from: https://imagej.nih.gov/ij/) for more detailed analysis of the appearance of symbols or dots, unmasked by the clearing milk. Investigating this would actually be a very nice piece of project work for a student.

[16] As this experiment currently stands, orlistat is an inhibitor of human pancreatic lipase (IC50 140 µM). The experiment as is, aims to get 140 µM approximately in the centre of any inhibition curve produced. However this has not been tested out on pig pancreatic lipase as yet. When it is we will publish the data here.

[17] See https://www.tes.com/lessons/P8AVsWCbzEvy2w/an-investigation-into-the-inhibition-of-milkfat-digestion-by-lipase. To see the full spreadsheet a number of columns need to be unhidden.

Enter your enquiry here

Fill in your details below or click an icon to log in:

WordPress.com Logo

You are commenting using your WordPress.com account. Log Out / Change )

Twitter picture

You are commenting using your Twitter account. Log Out / Change )

Facebook photo

You are commenting using your Facebook account. Log Out / Change )

Google+ photo

You are commenting using your Google+ account. Log Out / Change )

Connecting to %s