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Biochemistry term2 Q&ATerm 2: Lipids
1) What are lipids?
Chemically heterogeneous compounds that are Insoluble in water and Soluble in organic
solvents.
2) What are the functions of lipids?
Structural: Provide barriers between the organism and the environment
Energy storage
Signalling messenger
3) What is lipid extraction?
Separating the lipids from other components of the sample.
a. Unbiased
b. Free from non-lipid material
4) What techniques are used in lipid extraction?
Organism or tissue specific.
Method using ability of lipids to dissolve in organic solvents.
Commonly two solvents used with one polar
Lipids are sensitive to rapid degradation through oxidation from solvents, oxygen and
enzymes
5) Explain the extraction methods?
Bligh - dyer method
Add chloroform and methanol mixture to the sample
Homogenise sample using vortex-mixing
Additional chloroform and methanol added to give two phases
Transfer bottom phase in new tube and wash
The process can be repeated to increase purity
Evaporate to dryness in nitrogen gas
Re-dissolve in solvent with antioxidant and store in -80 oC
Changes in pH and solvent can be used to extract specific classes
Soxhlet extraction
Solid phase sample in container
Solvent evaporates when heated
Solvent becomes liquid in the condenser and drops in the sample container
When solvent overflows sample container it is emptied back to the solvent flask
After few hours the dissolved sample has been extracted in the solvent flask
6) Why do we need to separate lipids?
Solvent based extraction is not perfect
Other non-lipid hydrophobic molecules
A mix of different lipid compounds
Separation of the mix into components according to their properties: Storage,
Identification, Measurement
7) What are some other methods used to separate lipids?
Thin layer absorption chromatography
, High-performance liquid chromatography
Gas chromatography
How this works
Chromatographic techniques
Distribution of between two phases
Moving phase (solvent)
Stationary phase (absorbent surface or column)
8) How do we detect lipids after separation?
Using mass spectrometry
Oleic acid
Nuclear magnetic resonance
spectroscopy
9) How do we deal with lipid detection computing and analysis?
Dealing with noise in the data (batch and systematic effects)
Filtering
Identifying specific patterns in a complex profile
Peak detection
Large databases storing standard spectra for known compounds (proprietary
libraries)
Alignment of spectral data
Quantification (relative or absolute)
Normalisation
10) How do we measure lipids in clinic?
Focus on a small number of lipids and lipoproteins relevant to health
Total cholesterol
Triglycerides
, High density lipoprotein cholesterol (HDL)
Anything missing???
Low density lipoprotein
Commonly estimated through the Friedewald formula than measured
LDL_cholesterol = Total_cholesterol – HDL_cholesterol – (Triglyceride/2.19)
for mmol/L
Relatively accurate when triglycerides is used > 4.5mmol/L
Total cholesterol
Based on the enzymatic hydrolysis of cholesterol esters in serum
The absorbance of light by the oxidised dye is divided by the absorbance of a
standard sample and multiplied by a constant
Triglycerides
Similar to total cholesterol the aim is to use the released peroxide to oxidise a die
High density lipoprotein
Use a blocking reagent that targets the apolipoprotein B containing lipoproteins
(VLDL + LDL) rendering them non-reactive to the cholesterol enzymes
α-cyclodextrin
Mg+2
dextran SO4
Follow the exact same procedure as for total cholesterol
NMR and MS can measure all lipoproteins and triglycerides directly
Lipid metabolism
1) Explain lipid metabolism?
Lipids metabolism is the use of lipids to obtain or store energy
2) How are lipids transported?
Lipids are transported through the circulatory system
Fatty acids bound to Albumin
Others are transported via lipoproteins
3) What are lipoproteins?
, Complexes of lipids with proteins withTriglyceride and cholesteryl ester core, Surface layer
of phospholipids, cholesterol and apolipoproteins
4) How are lipoproteins classified?
They are classified by density and size
5) What is the difference between the
exogenous pathway and endogenous
pathway?
In the endogenous path- way,
cholesterol is synthesized by the liver
and extra- hepatic tissues and secreted
into plasma, whereas the intestine is the
primary site of the exogenous pathway
of dietary cholesterol uptake
6) What are chylomicrons?
Chylomicrons are large triglyceride-
rich lipoproteins produced in
enterocytes from dietary lipids—namely,
fatty acids, and
cholesterol. Chylomicrons are composed
of a main central lipid core that consists
primarily of triglycerides, however like
other lipoproteins, they carry esterified cholesterol and phospholipids (EXOGENOUS
PATHWAY)
7) Explain Vey low density lipoproteins (VLDL)?
VLDL is one of the three main types of lipoproteins. VLDL contains the highest amount of
triglycerides. VLDL is a type of "bad cholesterol" because it helps cholesterol build up on the
walls of arteries
8) Explain Low density lipoprotein (LDL)?
LDL is sometimes called the "bad" cholesterol because a high LDL level leads to a build-up of
cholesterol in your arteries.
If you have a high LDL level, this means that you have too much LDL cholesterol in your
blood. This extra LDL, along with other substances, forms plaque. The plaque builds up in
your arteries causing coronary artery disease.
9) Explain High density lipoprotein (HDL)?
HDL It is sometimes called the "good" cholesterol because it carries cholesterol from other
parts of your body back to your liver. Your liver then removes the cholesterol from your
body.
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