BIOLS 315

Biochemistry Lab. No.8

Lipid Metabolism

Materials

50 mM Tris-HCl, pH 7.5-8 250 ml

0.07 M CaCl2 300 ml
Phenolphthalein Indicator -
5 M NaOH 50 ml
5 M HCl 50 ml
2% Artificial Bile Salt 30 ml
Lipase, Pancreas (1mg/ml) 5 ml Freshly prepared
Lipase, Pancreas (0.5mg/ml) 5 ml Freshly prepared
Development system Prepare before use
Diethyl ether
10% phosphomolybdic acid 30-50 ml

Procedure
1. To 3 ml artificial bile salt solution add 5 drops of sunflower oil in a large test tube.
2. Warm the mixture in a water bath (boiling) with vigorous shaking until getting a
stable emulsion.
3. Cool the emulsion with shaking.
4. Add the following to the emulsion:

5 ml 0.05 M Tris-HCl
7 ml 0.05 M CaCl2 solution
Few drops of phenolphthalein indicator solution.

5. Place the mixture in a water bath at 37C and allow equilibrating (4-5 min).
6. Add 1 ml of lipase (keep the enzyme on ice) solution to the emulsion and mix.
7. Add few drops of 5 M NaOH until you get a pink color.
8. Immediately transfer 1 ml into a micro test tube and add 2 drops of 5 M HCl (zero
time).
9. At 0, 5, 10, 20 and 40 minutes take 1 ml sample and treat as above (add 2 drops of
5 M HCl, step no. 8).

(During the incubation period shake the mixture regularly and maintain the pH
at 8 by adding one drop of 5 M NaOH to maintain the pink color)

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10. To each of the withdrawn sample add 1 ml of diethyl ether, shake very well, and
allow the mixture to separate into phases.
11. Using micro capillary tubes, withdraw samples from the upper ether phase and
spot on a silica gel TLC (2 times).
12. Develop the TLC plate using petroleum ether: ether: glacial acetic acid (80:20:1)
Total volume = 10 ml each group or 100ml for all groups in 1 container. (Note:
Development may take 15 min).
13. Dry the plate and spray with 10% phosphomolybdic acid.
14. Put the TLC in the oven at 90C for 10 min.

Assignment

1. Bile salt solution was added to sunflower oil and the mixture
was incubated at 37oC. Explain the importance of this step in
lipids metabolism.
2. On what basis did the separation of different lipids by the use of
TLC technique was done?
3. The TLC plate was developed using three types of solvents
(petroleum ether, ether, and glacial acetic acid). Explain the
reason behind using more than one solvent.
4. Complete the following table by briefly describing the function
of each reagent or chemical used in this experiment.
5. Write the references that you have used to find out the
information gathered for this assignment.

Chemical Function or purpose

Tris-HCl
CaCl2
NaOH
HCl
Diethyl ether
Phenolphthalein

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List the references that you have used to find out this information
following this style:

References

Mayo Clinic. (2019). How eating fish helps your heart. Available at
https://www.mayoclinic.org/diseases-conditions/heart-disease/in-
depth/omega-3/art-20045614.

Lindshield, B. (2018). Kansas State University Human Nutrition (FNDH

400) Flexbook. 1st edition. Manhattan, New York City: New Prairie
Press, pp. 103.

Smith, S. (2009). Mechanism of chain length determination in

biosynthesis of milk fatty acids. Journal of Mammary Gland Biology and
Neoplasia, 14(3), 245-260.

Samiee, K., Rustaiyan. A (2015). Omega-3 Fatty Acids Composition and

Lipid Content from Liver and Muscle Tissues of Scomberomorus
commerson (Lacepede, 1800) in the Persian Gulf. Journal of Food and
Nutrition Research 3(9):620-623. doi: 10.12691/jfnr-3-9-10.

Geleijnse, J. M., Giltay, E. J., Grobbee, D. E., Donders, A. R., Kok, F. J.

(2002). Blood pressure response to fish oil supplementation:
metaregression analysis of randomized trials. Journal of
hypertension, 20(8), 1493–1499.