Lab 8 Lipid Extraction and Oxidation

Question 1

Describe Goldfisch fat extraction.

Answer 1

• Requires that food be dried
• Polar solvents (water) will interfere with the extraction in the presence of highly non-polar solvent like petroleum ether (mixture of short hydrocarbons)
• Extracts non-polar lipids

Question 2

Describe the Bligh-Dyer method.

Answer 2

• Fresh (wet) sample is used
• Methanol helps extract polar lipids
• Chloroform helps extract non-polar lipids

Question 3

After extraction via the Bligh-Dyer method, where are the lipids concentrated?

Answer 3

In the chloroform layer.

Question 4

How is oxidation initiated in living cells?

Answer 4

• By reactive oxygen species (ROS)
• ROS are naturally found in all cells as they are intermediates in mitochondrial processes and enzymatic reactions

Question 5

How can the extent of oxidation be measured, if not by iodometric titration to determine primary oxidation products?

Answer 5

• Malondialdehyde (MDA) is a by-product of lipid oxidation
  
  • Peroxyl radical formed via ROS
  • Molecule temporarily becomes cyclic
  • Lipid fragments, yielding MDA
• MDA can be quantified by reacting with thiobarbituric acid (TBA) to produce a red compound

Question 6

How is TBARS quantified?

Answer 6

• Beer’s Law

Question 7

What are lipids?

Answer 7

Organic compounds that are insoluble in water but soluble in organic solvents.

Question 8

Describe the two general types of lipids.

Answer 8

• Triacylglycerols which contain hydrolysable ester linkages
• Other lipids are sterols which do not have ester linkages and therefore cannot undergo hydrolysis or saponification.

Question 9

When are lipids solid/liquid at room temperature?

Answer 9

• Fats are solid at room temperature and contain predominantly saturated fatty acids.
• Oils are liquid at room temperature and contain predominantly unsaturated fatty acids.

Question 10

How are fats and oils classified?

Answer 10

• According to chain length, position and configuration of double bonds, and by the occurrence of additional functional groups

Question 11

What is a critical factor affecting tissue stability during frozen storage of fish?

Answer 11

• Lipid content
• Fatty fish confer high nutritional value, but the lipid content increases susceptibility to oxidative rancidity.

Question 12

Describe the TBARS assay.

Answer 12

• This assay uses thiobarbituric acid (TBA) to measure malondialdehyde, a secondary product of lipid oxidation.
• The TBA is heated with an extract obtained from an acidified fish sample, forming a red pigment and the colour intensity is proportional to the malondialdehyde content.

Question 13

In the Bligh Dyer lipid extraction method, which layer is the chloroform in the biphasic solution in the graduated cylinder?

Answer 13

Bottom layer

Question 14

What is the purpose of the hot plate in the Bligh Dyer lipid extraction method?

Answer 14

To evaporate chloroform.

Question 15

Which solvent is used in the Goldfisch fat extraction method?

Answer 15

Petroleum ether

Question 16

The TBARS assay requires a standard curve for quantification
True or False?

Answer 16

False.

TBARs uses Beer’s Law.

Question 17

In the TBARS assay, after centrifuging, which layer is vacuum filtered?

Answer 17

Aqueous layer

This is after centrifuging for 10 minutes at 3000 g and disgarding the hexane layer (top layer).

Question 18

In the TBARS assay, what solvent can you use to rinse your stomacher bag when quantitatively transferring?

Answer 18

Hexane

Question 19

Which fats cannot undergo hydrolysis or saponification?

Answer 19

Those without ester linkages (like sterols)

Question 20

What does hydrolysis of a fat with an alkali yield?

Answer 20

Glycerol and free fatty acids

Question 21

What is the purpose of the trichloroacetic acid in the TBARS oxidation quantification?

Answer 21

In the TBARs assay, trichloroacetic acid (TCA) is used to precipitate macromolecules and accelerate the condensation reaction of the reagent TBA with malondialdehyde (MDA), since TCA is a stronger acid compared with acetic acid. This results in more color of the chromophore (red) being produced.

Question 22

What is the purpose of the butylated hydroxytoluene (BHT) in hexane in the TBARS assay?

Answer 22

Butylated hydroxytoluene (BHT) is used in the lysis buffer during sample preparation to stop further sample peroxidation while processing.

Question 23

After adding chloroform and methanol to the fresh fish sample in a stomacher bag and blending for 2 minutes, what is the purpose of adding saturated NaCl?

Answer 23

This helps prevent an emulsion of the two solvents and thereby stabilize the biphasic solution for a clearer separation.