LIPIDS
BIOCHEMISTRY
LABORATORY
by: JEAN C. APLASCA, RPh
OBJECTIVES
• To become acquainted with the general
properties of lipids and fatty acids
• To understand the classification of lipids
• To distingush between fats and oils
• To tabulate General and specifc tests for lipids,
including their positive results
GENERAL PROPERTIES
• contain C,H2,O2
• exist as: fats, oils, cholesterols, hormones
• organic compounds formed mainly from
alcohol and fatty acids combined together by
ester linkage.
• insoluble in water, but soluble in organic
solvents
• not a polymer
CLASSIFICATION OF
LIPIDS
1. SIMPLE LIPIDS
• Esters of fatty acids with glycerol. (Eg. Fats and waxes)
• The triacylglycerol (TAG) is the simplest and most common fat. It is the
form in which lipids are stored in the cell. Also known as neutral fat.
2. COMPOUND LIPIDS (Conjugated lipids)
• yield one or more FA, an alcohol, and other type of compounds
(proteolipids, phospholipids & glycolipids)
• Glycerol, Fatty acid, Nitrogenous base and Phosphoric acid.
3. PRECURSOR AND DERIVED LIPIDS
• Precursor lipids are compounds produced when simple and complex
lipids undergo hydrolysis. (fatty acids, glycerol, sphingosine, and other
alcohols)
• Derived lipids are formed by metabolic transformation of fatty acids.
(ketone bodies, steroids, fatty aldehydes, prostaglandins and lipid-
soluble vitamins)
TWO COMMON METHODS
IN SUBCLASSIFYING
LIPIDS
A. Based on biochemical function
1. Energy-storage lipids
‒ TAG
[Link] lipids
‒ phospholipids
‒ sphingoglycolipids
‒ cholesterol
3. Emulsification lipids
‒ bile acids
4. Messenger lipids
‒ steroid hormones
‒ eicosanoids
5. Protective-coating lipids
‒ biological waxes
B. Based upon whether or not saponification occurs
when a lipid is placed in basic aqueous solution
1. Saponifiable lipids converted into smaller
‒ TAG molecules when hydrolysis
‒ phospholipids occurs
‒ sphingoglycolipids
‒ biological waxes
2. Nonsaponifiable lipids cannot be broken up into
‒ cholesterol smaller units since they do
‒ steroid hormones not react with water
‒ bile acids
‒ eicosanoids
ENERGY-STORAGE
LIPIDS
TRIGLYCERIDES
“Triacylglycerol”
– a lipid formed by esterification of 3 fatty acids to
a glycerol molecule
GLYCEROL
– a trihydric alcohol and has the popular name
glycerin.
– synthesized in the body from glucose
– colorless viscous oily liquid with sweet taste
– trinitroglycerin (TNT) is used as explosive and
vasodilator
FATTY ACIDS
• carboxylic acids involved in TAG formation
• Fatty acids differ from one another in:
– length of the hydrocarbon tails
– degree of unsaturation (double bond)
– position of the double bonds in the chain
TYPES OF
FATTY ACIDS
SATURATED FATTY ACIDS
• All C bonded to H
• No C=C double bonds
– long, straight chain
– solid at room temp except if they are short
chained
– mostly animals fats
– contributes to cardiovascular diseases
(atherosclerosis)
UNSATURATED FATTY ACIDS
• C=C double bonds in the fatty acids (“cis”)
– plant & fish fats
– vegetable oils
– liquid at room temperature
– the kinks made by double bonded C prevent the
molecules from packing tightly together
mono-unsaturated?
poly-unsaturated?
Saturated vs.
Unsaturated
FATS AND OILS
• naturally occuring mixtures of TAG molecules in which there
are many different kinds of TAG molecules present.
• colorless, odorless, tasteless
FATS OILS
- solid or semi-solid at room - liquid at room temperature
temp (25°c) (25°c)
- animal source - plant source
- saturated FA predominate - mono & polyunsaturated FA
- “linearity” - “bends”
- higher melting points - lower melting points
SAPONIFICATION
• a process by which triglycerides are reacted with
sodium or potassium hydroxide (lye) to produce
glycerol and a fatty acid salt, called “soap”.
• most often animal fats or vegetable oils
sodium hydroxide = hard soap
potassium hydroxide = soft soap
Alkaline hydrolysis of esters
SALTING OUT OF SOAP
• purification method that utilizes the
reduced solubility of certain molecules in a
solution of very high ionic strength.
• Soap is precipitated as a solid from the
suspension by adding common salt to the
suspension.
TEST FOR UNSATURATION
Principle:
The most important application of the iodine value is
to determine the amount of unsaturation contained
in fatty acids. This unsaturation is in the form of
double bonds which react with iodine compounds.
The higher the iodine value, the more unsaturated
fatty acid bonds are present in a fat.
Brain lipids
- Lipids are used by living organisms such as
humans for energy storage and can also be found
in different parts of the body, especially the brain.
Pig’s brain contains a high amount of lipid content,
the docosahexanoic acid (DHA) or popularly known
as omega-3 fatty acid. Different complex lipids that
can be found in Pig’s brain are complex lipids such
as Cholesterol, Phosphatides, Cerebrosides and
Sphingosines
TEST TEST FOR (PRESENCE) POSITIVE RESULT
SAPONIFICATION ester bonds bubbles & precipitate
double bond, degree of colorisation of sol'n (yellow
UNSATURATION
saturation to orange)
SALKOWSKI cholesterol yellow to brick red
LIEBERMANN-BURCHARD cholesterol deep green
MOLISCH carbohydrates purple ring
KRAUT'S choline (lecithin) red orange
free amino groups & secondary
NINHYDRIN blue-violet
amines
burnt grease smell &
ACROLEIN glycerol/lipids
blackening
SUDAN IV glycerol/lipids red sol'n
Appearance of Isolated Complex Lipid Samples
Sample Appearance of Filtrate
Cholesterol Clear colorless solution
Glycerophosphatides Brownish yellow solution
Sphingosine Phosphatides and Slightly turbid with suspended
Sphingosine Glycosides precipitate
SALKOWSKI TEST
A yellow to brick-red
colour is formed
indicating the presence
of cholesterol.
There may be a red color
in the upper layer
(chloroform) and green
fluorescence in the lower
layer due to the heavier
sulfuric acid settling.
Liebermann-Burchard test
• DEEP GREEN COLOR
• It is used to detect the presence of
steroids. This color is due to the OH
group of cholesterol and the
unsaturation found in the adjacent
fused ring.
• The color change is gradual: first it
appears as a pink coloration,
changing later to lilac, and finally to
deep green.
• the intensity is roughly proportional
to the amount of steroid present
MOLISCH TEST
A purple ring is
formed indicating the
presence of
carbohydrates.
KRAUT'S TEST
• Red- orange solution
• Kraut’s test is a specific test for
Choline (Lecithin) such as
glycerophosphatides,
sphingolipids and
phosphorylated lipids
NINHYDRIN TEST
• Blue- violet solution
indicates a positive
result
• Test mainly used for
cephalins, lecithins,
and sphingomyelins
ACROLEIN TEST
• is used to detect the presence of glycerol or fat
• Note:When fat is treated strongly in the presence of
a dehydrating agent like potassium bisulphate
(KHSO4), the glycerol portion of the molecule is
dehydrated to form an unsaturated aldehyde,
acrolein that has a pungent irritating odour.
• positive result:
– irritating acrid smell and a burnt grease
MEMBRANE
LIPIDS
1. PHOSPHOLIPIDS
a lipid that contains one or more fatty acids, a
phosphate group, a platform molecule to which
the fatty acid(s) and the phosphate group are
attached, and an alcohol that is attached to the
phosphate group.
• Glycerophospholipid = glycerol + 2 FA + PO4
• Sphingophospholipid = sphingosine + 1 FA + PO4
2. SPHINGOGLYCOLIPIDS
a lipid that contains both a fatty acid and a
carbohydrate component attached to a
sphingosine molecule
Cerebrosides
• single monosaccharide unit (glucose or galactose)
• occur primarily in the brain (7% of dry mass)
SPHINGOSINE
– the alcohol (monohydric) present in
sphingolipids
– synthesized in the body from serine and
palmitic acid
– not positive with acrolein test
3. CHOLESTEROL
• a C27 steroid molecule
that is a component of
cell membranes and a
precursor for other
steroid-based lipids
• high levels in blood may
contribute to
cardiovascular disease
STEROIDS
a lipid whose structure is based on a fused ring system that
involves three 6-membered rings and one 5-membered ring
structure:
4 fused C rings + ?
- different steroids created by attaching different functional groups to
rings
- different structure creates different function
examples:
cholesterol, sex hormones
SEX HORMONES
Review Questions
1. Lipid molecule hydrolysis produces
A. Glycerol and fatty acids
B. Glycerol and water
C. Water and amino acids
D. Glucose and fatty acids
E. Water and fatty acids
2. In phospholipids, at least one fatty acid chain is
“kinked”, resulting in a bent structure. This
phenomenon, which gives fluidity to cell
membranes, is caused by
A. Excess hydrogen atoms around the bond
B. Hydrophobic interactions
C. Multiple double bonds
D. Sulfhydryl group interactions
E. Multiple ionic bonds
3. The single structural unit common to all
lipids is
A. Fat
B. Glycogen
C. Cholesterol
D. Glycerol
E. Carbon
