BIOCHEMISTRY FOR MEDICAL LABORATORY SCIENCE BSMT 1

LABORATORY| JAN CLARENCE SALAS 2nd Semester

MTCHEM2 - MODULE 3: EXPERIMENT 5 – LIPIDS

SATURATED FATTY UNSATURATED FATTY

OUTLINE ACIDS ACIDS

I. Introduction C-C double bonds are

Linked by single C-C bonds present
II. Lipids
III. Classification of Lipids
Higher melting points Low melting point
IV. Fats and Oils
Liquid at room temperature Monounsaturated Fatty
solid at cold temp. Acid, Polyunsaturated Fatty
INTRODUCTION Acid

Lipids are naturally occurring substances that are grouped

SFA>UFA = Higher Melting Point
together on the basis of their solubility in non-polar solvents like
chloroform, carbon tetrachloride and ether, and their insolubility
• As the number of the double bond in the fatty acid
in water. They play important roles in the structure of cell
increases, the melting point decreases
membranes and serve as major sources of energy in animals
and plants. • The longer the hydrocarbon chain, the higher the melting
point.
Lipids maybe classified based on their major roles in the body:
storage, membrane component and chemical messengers. CLASSIFICATION OF LIPIDS
Storage lipids include triglycerides which are divided into fats
and oils. They are triesters of glycerol and fatty acids. They are ACCORDING TO FUNCTION
significant components of fat storage cells.
• Energy Storage Lipids
Membrane component lipids are usually amphipathic and o Intracellular storage of metabolic fuel such as
complex. They are classified into two groups: phospholipids and triacylglycerols.
glycolipids. Cell membranes are made of a lipid bilayer • Membrane Lipids
consisting of phospholipids. o structural components of cell membranes such as
phospholipids, sphingoglycolipids, and cholesterol.
The third group of lipids are the chemical messengers. These • Emulsification Lipids
include steroids characterized by the presence of a fused four- o lipids that stabilize and disperse water insoluble
ring nucleus. The most common steroid is cholesterol and materials in aqueous solution such as bile acids.
serves as a starting material for the formation of hormones and • Messenger Lipids
bile salts. o regulatory lipids that act in the tissues where they are
synthesized after transferred via bloodstream such as
LIPIDS steroid hormones and eicosanoids.
• Protective-Coating Lipids
• Comes from a Greek word lipos meaning “fat”or “lard” o water insoluble/water repellent lipids with protective
• An organic compound found in living organisms that is coating and lubricant functions as biological waxes.
insoluble (or sparingly soluble) in water but soluble in non-
polar organic solvents such as chloroform, ether, and ACCORDING TO HYDROLYSIS
benzene.
• Saponifiable
Non-Polar Solvents Polar Solvents • Non-Saponifiable
Lipophilic as they dissolve
FATS AND OILS
non-polar substances such Hydrophilic as they are
as oils, fats, and greases miscible with water
Are the most abundant lipids in nature; they provide energy for
living organisms, insulate body organs, and transfer fat soluble
Examples: Examples:
vitamins through the blood.
1. Chloroform 1. Water
2. Carbon 2. Acetic Acid
FATS OILS
Tetrachloride 3. Methanol
3. Methylene Chloride 4. Ethanol • triglycerides • triglycerides
4. Diethyl Ether 5. n-propanol • typically obtained from • typically obtained from
5. Benzene animals plants
• solids at room • liquids at room
temperature temperature
FATTY ACIDS

• one of the building blocks of simple and compound lipids

• monocarboxylic acids with hydrocarbon residues that are
acyclic and unbranched

DIMATULAC | BSMT 1-A 1

 TRIGLYCERIDES SOLUBILITY TEST

• Major storage form of energy in the body PRINCIPLE: Lipids are soluble in non-polar substances but non-
• Most abundant dietary lipid: margarine, butter, cooking oils soluble in polar substances.
• Physiologic calorie equivalent of one gram of the
o Fat: 9 Kcal Solubility of coconut oil and margarine
o Carbohydrates: 4Kcal 1. hexane
o Protein: 4 Kcal 2. water
• Carbohydrate is still the primary energy source 3. ethyl alcohol
• Ester of fatty acids with glycerol 4. HCl
• A simple lipid: composed of an alcohol backbone and fatty 5. NaOH
acids
Ethyl HCl Sodium
Samples Hexane Water
Alcohol Acid Hydroxide
MEMBRANE LIPIDS Coconut
Miscible Immiscible Immiscible Immiscible Immiscible
Oil
Complex lipids composed of esters of fatty acids and various
alcohol groups or functional groups Margarine Soluble Insoluble Insoluble Insoluble Insoluble

Phospholipids contain a phosphate group, glycerol, and fatty

acids; major component of the cell membrane. TEST FOR UNSATURATED GLYCERIDES

Glycolipids contain a carbohydrate and fatty acid chains; PRINCIPLE: Iodine is taken up by the double C=C bonds in
maintain stability of the membrane, act as receptors unsaturated fat.

Therefore, the more unsaturated, more double bonds, more

LIPIDS AS CHEMICAL MESSENGERS
iodine is taken up, the clearer the liquid.
Steroids: a biologically active organic compound with four rings
Quantitatively measured as the iodine number: grams of iodine
arranged in a specific molecular configuration.
absorbed by 100 grams of the substance.
Two functions: components of cell membranes that alter
Saturated fats have an iodine number of zero.
membrane fluidity; and as signaling molecules
3 drops of alcoholic iodine
Cholesterol: Cyclopentanoperhydrophenanthrene ring
SAMPLES RESULT INTERPRETATION
CHOLESTEROL
Color Change Less Unsaturated
Coconut Oil
Lighter Color Fatty Acids
• Precursor of all steroid hormones
o sex hormones Color Change More Unsaturated
Cottonseed Oil
o cortisol Lightest Color Fatty Acids
o aldosterone
• Precursor of vitamin D3 SAPONIFICATION
o absorption of calcium and phosphorus
• Precursor of bile acids PRINCIPLE: Hydrolysis of fats or triglycerides by combining
o absorption of fat-soluble vitamins A, D, E, K them with a strong alkali in order to create glycerol and
• Good in moderation, bad in excess and deficiency potassium salts of them.

PHYSICAL DETERMINATION OF FATS AND OILS Lipids with fatty acids are saponifiable, while those without are
not.
SPOTTING EFFECT: PRACTICAL TEST FOR LIPIDS

PRINCIPLE: Water has a lower boiling point, so it will evaporate

more quickly than lipids. Lipids will remain penetrated onto the
filter paper and diffract light.

Diffraction: occurs when light waves pass near a barrier and

spread out

Oils with shorter fatty acid chains or unsaturated will disappear

more quickly because they have lower boiling points

SAMPLES RESULT
Ethyl Alcohol Absence of Translucent Spot First Test Tube
Acetic Acid Absence of Translucent Spot 1. 4 drops of vegetable oil
2. 10 drops of 10% NaOH solution
Coconut Oil Presence of Translucent Spot 3. 5 mL distilled water
Margarine Presence of Translucent Spot 4. HEAT THE CONTENTS – LOW FLAME

DIMATULAC | BSMT 1-A 2

Second Test Tube
1. 4 drops of vegetable oil
2. 10 drops of 10% NaOH solution
3. 5 mL distilled water
4. DO NOT HEAT

1ST TEST TUBE (+) 2ND TEST TUBE (-)

Formation of white bubbles No formation of white bubbles
Unclear Liquid Slightly clear liquid

DETECTION OF CHOLESTEROL IN FOOD SAMPLES

SALKOSWKI TEST

Positive result: two layers; chloroform layer has bluish

red/violet color, while the sulfuric acid layer will have a
yellow/green color

PRINCIPLE: sulfuric acid dehydrates cholesterol, which forms

Cholestadiene. Cholestadiene dimerizes and is sulfonated,
giving rise to the color change.

1. 1g of canned meat, dissolve in 2mL chloroform

2. 2mL concentrated sulfuric acid

STEPS:

1. Dehydration of Cholesterol - Formation of

Cholestadiene
2. Dimerization of Cholestadiene – Formation of bi-
Cholestadiene
3. Sulfonation of bi-Cholestadiene

LIEBERMANN-BURHCARD TEST

Positive result: blue/green color

PRINCIPLE: sulfuric acid and acetic anhydride in chloroform

rapidly converts cholesterol to its acetate and sulfate
derivatives, which are further sulfonated. Once sulfonated, the
blue/green color is formed

1. 1g of canned meat, dissolve in 2mL chloroform

2. 10 drops acetic anhydride
3. 2 drops concentrated sulfuric acid

COMPONENTS

1. Sulfate Derivative of Cholesterol

2. Acetate Derivative of Cholesterol

SAMPLE RESULT INTERPRETATION

Red-Brown
Salkowski Test Positive
Precipitate

Libermann- Green or Green-

Positive
Burchard Test Blue Color

DIMATULAC | BSMT 1-A 3