2.

Natural Chemistry of Lipids

DE S TA S E YO UM (C.PH AR M A CIS T )

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 Lipids are broadly defined as any fat-soluble (lipophilic),
naturally-occurring molecule, such as fats, oils, waxes,
cholesterol, sterols, fat-soluble vitamins (such as vitamins
A, D, E and K), monoglycerides, diglycerides,
phospholipids, and others.
The main biological functions of lipids include energy
storage, acting as structural components of cell
membranes, and participating as important signaling
molecules.
Although the term lipid is sometimes used as a synonym
for fats, fats are a subgroup of lipids called triglycerides.
Lipids also encompass molecules such as fatty acids and
their derivatives (including tri-, di-, and monoglycerides
and phospholipids), as well as other sterol-containing
metabolites such as cholesterol.
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 Classification of lipids
On the basis of the nature of the products obtained on
hydrolysis, Lipids are mainly divided in two types
such as simple Lipids and Compound Lipids.
Besides thus there is one more type called derived
lipids .
Simple lipids :- Lipids which upon Hydrolysis yield
one or more fatty acids and an alcohol.
Compound Lipids :- Lipids which upon Hydrolysis
yield in addition to fatty acids and alcohol , compound
such as phosphoric acid , sugar ,etc

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Derived Lipids:- Lipids which also do not contain any
ester linkage but may be considered to have been derived
from naturally occurring esterified materials .
 Most of this has been alcohols of complex nature. eg.
Certain steroids, Certain Vitamins ,and certain plant
pigments .

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Lipids may be broadly defined as hydrophobic or
amphiphilic small molecules that originate
entirely or in part from two distinct types of
biochemical subunits or "building blocks":
ketoacyl and isoprene groups.
Using this approach, lipids may be divided into
eight categories : fatty acyls, glycerolipids,
glycerophospholipids, sphingolipids,
saccharolipids and polyketides (derived from
condensation of ketoacyl subunits); and sterol
lipids and prenol lipids (derived from
condensation of isoprene subunits)

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 Categories of Lipids
7

1 Fatty acyls
2 Glycerolipids
3 Glycerophospholipids
4 Sphingolipids
5 Saccharolipids
6 Polyketides
7 Sterol lipids
8 Prenol lipids

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 8

1. Fatty acyls
Fatty acyls (including fatty acids) are a diverse group of
molecules synthesized by chain-elongation of an acetyl-
CoA primer with malonyl-CoA or methylmalonyl-CoA
groups.
The fatty acyl structure represents the major lipid
building block of complex lipids and therefore is one of
the most fundamental categories of biological lipids.

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 9

The carbon chain may be saturated or unsaturated, and

may be attached to functional groups containing
oxygen, halogens, nitrogen and sulfur.
Examples of biologically interesting fatty acyls are the
eicosanoids which are in turn derived primarily from
arachidonic acid and eicosapentaenoic acid, which
include prostaglandins, leukotrienes, and thromboxanes.

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 10

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 11

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 12

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 13

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 Triglycerides
14

Triglyceride: an ester of glycerol with three fatty acids

O
O CH 2 OCR
1 . NaOH, H 2O
R'COCH O
2 . HCl, H 2O
CH 2 OCR''
A triacylglycerol CH 2 OH RCO2 H
(a triglyceride)
HOCH + R'CO 2 H
CH 2 OH R''CO 2 H
1,2,3-Propanetriol Fatty acids
(Glycerol, glycerin)
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 15

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 16

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 17

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 2. Glycerolipids
Glycerolipids are composed mainly of mono-, di- and
tri-substituted glycerols , the most well-known being
the fatty acid esters of glycerol (triacylglycerols), also
known as triglycerides.

These comprise the bulk of storage fat in animal

tissues.

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Glycerolipids are complex lipids formed by the
condensation of one, two, or three fatty acid molecules on
glycerol, a small compound with three carbon atoms (either
numerically numbered C1, C2, C3, or, according to the Greek
alphabet, Cα, Cβ, Cα’), with each one bearing a hydroxyl
function OH. Glycerol is a symmetrical molecule and its two
terminal –CH2OH groups (referred to as α and α’) are
stereochemically equivalent. The central carbon C2, which is
not asymmetric, is referred to as β.

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 Glycerolipids Synthesis

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Each OH function of glycerol can react with the –COOH group
of a fatty acid, leading to an ester derivative called glyceride.
An ester results from the condensation of a carboxylic acid and
an alcohol.

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Each carbon atom of glycerol can be linked to a fatty acid,
which will then become a chemical group by itself referred to
as “acyl.” If all three carbons of glycerol have reacted with a
fatty acid, we will obtain a triacylglycerol (TAG), also named
triglyceride. Diacylglycerol (DAG, diglycerides) include either
αα’ or αβ derivatives, according to the site of esterification.5
Similarly, the two categories of monoacylglycerol (MAG,
monoglycerides) are α and β. Examples of MAG, DAG, and
TAG (mono-, di-, or triacylglycerol) structures are given in
Fig. 1.12.

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Most membrane glycerolipids are phospholipids. These
membrane components are obtained by the condensation of
an αβ-DAG with phosphoric acid, leading to a molecule called
phosphatidic acid (PA). PA is the precursor of membrane
glycerophospholipids. The most important biochemical
feature of this class of compounds is the nature of the acyl
chain in α and β of glycerol; R1 (in position α) results from the
condensation of a saturated fatty acid, whereas R2 (in position
β) comes from an unsaturated fatty acid. This unique
combination of saturated/unsaturated chains is critical for
proper membrane function
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PA6 is a metabolic intermediate in the
biosynthetic/degradation pathways of more complex
glycerophospholipids. It usually represents less than 1% of
total membrane lipids but plays a critical role in signal
transduction, due to the unique ionization properties of its
phosphate group (see Chapter 3). Membrane
glycerophospholipids are derived from PA by condensation
with an organic alcohol (general formula X-OH).
Phosphatidylcholine (PC), the most abundant membrane
lipid, results from the condensation of choline with PA

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 Cardiolipin(Diphosphotidylglycerol

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 3. Glycerophospholipids
Glycerophospholipids, also referred to as phospholipids,
are ubiquitous in nature and are key components of the
lipid bilayer of cells, as well as being involved in
metabolism and signaling.

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 Glycerophospholipids, also referred to as phospholipids, are
ubiquitous in nature and are key components of the lipid bilayer of
cells, as well as being involved in metabolism and signaling.
Glycerophospholipids may be subdivided into distinct classes,
based on the nature of the polar head group at the sn-3 position of
the glycerol backbone in eukaryotes and eubacteria or the sn-1
position in the case of archaebacteria.
Examples of glycerophospholipids found in biological membranes
are phosphatidylcholine (also known as PC or GPCho, and
lecithin),phosphatidylethanolamine (PE or GPEtn) and
phosphatidylserine (PS or GPSer).
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  Phospholipids are the second most abundant
group of naturally occurring lipids
 they are found almost exclusively in plant and
animal membranes, which typically consist of 40% -
50% phospholipids and 50% - 60% proteins
 the most abundant phospholipids are derived from
phosphatidic acid, a molecule in which glycerol is
esterified with two molecules of fatty acid and one
of phosphoric acid
 the three most abundant fatty acids in phosphatidic
acids are palmitic acid (16:0), stearic acid (18:0),
and oleic acid (18:1)

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 Phospholipids

O
stearic acid CH2 -O-P-O -
O
-
CH O
O
O CH2
palmitic acid O glycerol

further esterification with a low-molecular

weight alcohol gives a phospholipid
among the most common of these low-molecular-
weight alcohols are

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 Phospholipids

Name and Formula Name of Phospholipid

ethanolamine
HOCH2 CH2 NH2 phosphatidylethanolamine
(cephalin)
choline +
HOCH2 CH2 N(CH 3 ) 3 phosphatidylcholine
(lecithin)
serine -
HOCH2 CHCO 2 phosphatidylserine
+
NH 3
inositol OH
HO HO OH phosphatidylinositol
HO OH

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 Phospholipids
choline
O
+
O P OCH 2 CH2 N(CH 3 ) 3
stearic acid O-
O CH2
O CH
O CH2 glycerol
O
palmitic acid

In aqueous solution, phospholipids spontaneously form into a

lipid bilayer, with a back-to-back arrangement of lipid monolayers

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In the Western diet, palmitic acid (C16:0) and stearic acid (C18:0)
are the most commonly consumed saturated fatty acids [2]. It is
generally believed that palmitic acid is more cholesterol-raising
than stearic acid.
The key difference between palmitic acid and stearic acid is that
palmitic acid is more cholesterol-raising than stearic acid.

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 Phosphotidylethanolamine

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 Phosphatidylcholine (PC)

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 • Structural Role:

• Role in blood coagulation:

– They are required at the stage of conversion of prothrombin

by active factor X

– activation of factor VIII by activated factor IX.

• Role in lipid absorption in intestine:

– Lecithin lowers the surface tension of water and aids in

emulsification of lipid water mixture,.

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 • Role in transport of lipids from intestines:

– Exogenous triglycerides is carried as lipoprotein complex,

chylomicrons, in which phospholipids takes an active part.

• Role in transport of lipids from liver:

• Role in electron transport:

• Lipotrophic action of Lecithin:

• Membrane phospholipids:

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 • Insulation:

– Phospholipids of myelin sheaths provide the insulation

around the nerve fibers.

• Role in Hormone action:

– provide communication between the hormone receptor on

the plasma membrane and intracellular Calcium reservoirs.

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 Role in PGs and leukotrienes

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 Selective inhibition by Aspirin

• Aspirin inhibits the production of PGI2 and TXA2

• PGI2-
– vasodilatation
– Decrease platelet aggregation
• TXA2-
– vasoconstriction
– increase platelet aggregation

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 • PGI2- Endothelium

• TXA2-Platelets

Irreversible inhibition of COX- present in platelets

Endothelial COX will be regenerated.

• In Low dose - less aspirin reach to peripheral tissue

compared to platelets.

• In High dose – do effect on both platelets as well as

endothelium
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 Role as second messenger

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 Role as second messenger

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 Lung surfactant
• Premature baby can suffer from ARDS(Acute Respiratory
Distress Syndrome)
• Following are Lung surfactant
– Dipalmitoyl-lecithin
– Sphingomyelin
• L to S ratio for lung maturity

• In premature babies, this surfactant is deficient and they suffer

from Respiratory Distress Syndrome.
• Glucocorticoids increase the synthesis of the surfactant
complex and promote differentiation of lung cells.

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Phospholipases are enzymes that hydrolyze specific portions of
phospholipid molecules. Their role in the digestion of exogenous
phospholipids and as the active principle in snake and bee venoms
has long been appreciated.

Classification of Phospholipases
Phospholipases are further classified into different types which are
presented in Tables 2 and 3 (mammalian PLA1/PLA2 and
PLC/PLD, respectively) and Table 4 (plant phospholipases). This
classification is based on their distribution, sequence similarity,
structure characteristics and requirements for optimum activity
in vitro, which is connected to the differential activation of these
enzymes.
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 Phospholipases are enzymes that degrade cell membranes to allow
pathogens to escape phagosomes.

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 Role of Phospholipase

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Snake bite cause severe haemolysis
The venom contains lecithinase,
hydrolyzes the PUFA converting lecithin into lysolecithin (detergent
like action).
Lysolecithin causes hemolysis of RBCs.
cause anaphylactic shock as well as bleeding tency.

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 4. Sphingolipids
Sphingolipids are a complex family of compounds that
share a common structural feature, a sphingoid base
backbone that is synthesized de novo from serine and a
long-chain fatty acyl CoA, then converted into
ceramides, phosphosphingolipids, glycosphingolipids
and other species.
The major sphingoid base of mammals is commonly
referred to as sphingosine.

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  Sphingolipids are a type of lipids made up of fatty acid chains
that were first mentioned in 1884 in J.L.W. Thudichum’s A Treatise
on the chemical composition of the brain.
 They were named after the Greek mythological creature, the
sphinx, due to the unknown riddle of their function.
 Sphingolipids are found in essentially all plants, animals, fungi
and in some prokaryotes and viruses. Specifically, they are found in
membranes and as a major component of lipoproteins.

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 Structure
 Sphingolipids are amphipathic molecules; they have hydrophobic
and hydrophilic properties.
 In the hydrophobic region there is a sphingoid long chain base
(aliphatic chains with attached hydroxyl groups) with a fatty acid
chain attached by amide bond at carbon 2.
 In the hydrophilic region, there are phosphate groups, sugar
residues, and/or hydroxyl groups. This amphipathic nature allows for
the diffusion of sphingolipids between membranes and for the
flipping of sphingolipids between membrane leaflets.

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However, sphingolipids are still more likely to accumulate in acidic
environments because of the possible ionization of a free amino
group.[4] There are several classes of sphingolipids: the sphingoid
base and simple derivatives, ceramides, and complex sphingolipids

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Sphingoid base & simple derivatives
 Sphingolipids are composed of many backbone “sphingoid
bases” which are synthesized from serine and a long-chain fatty
acyl-CoA. If a backbone has not yet been analyzed, it is referred to
as a sphinosine, after the original name for the fundamental
component of these lipids.
 Sphingoid bases can vary in alkyl chain length/branching, the
number and position of double bonds, the number and locations of
hydroxyl groups, etc.

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  These simple derivatives exist mainly as the backbone of more
complex sphingolipids, but they do exist on their own. They are
mainly used in intra-and extra-cellular signaling.
 The exact structure of the sphingolipid determines the function.
For example, in the dermis, there are additional hydroxyl groups at
positions 4 and/or 6 that interact with neighboring molecules to
strengthen the permeability of the skin barrier.

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 Ceramides
 Ceramides, which are synthesized in the endoplasmic reticulum,
are the simplest sphingolipid after the backbone.
 They are fatty acid derivatives of sphingoid bases, linked by amide
bonds. They are usually fully saturated or mono-unsaturated and
consist of 14-26 carbons, but are sometimes longer. There is
sometimes a hydroxyl group on the ω or ω-carbon atom.
 Ceramides have high phase transition temperatures of greater than
37°C. This is due to their predominately saturated nature, which
allows for the tight packing of lipids. This also favors the segregation
of ceramides (and more complex sphingolipids) into regions called
membrane
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 Ceramides are key to cell signaling processes. They generally
serve as second messengers by regulating cell growth, senescence,
and apoptosis.
 The exact function depends on the type of sphingolipid base and
fatty acid. For example, ceramides containing palmitic acid for a fatty
acid chain are generated in response to apoptosis.
 Much of the specifics of ceramide function is still under
investigation. Ceramides also serve as a precursor to more complex
sphingolipids.
 Ceramides can only exist in membranes but they have a rapid flip
rate between leaflets. This means they cannot leave the organelle they
are created in, but will have access to binding proteins and enzymes
on both sides of the bilayer. As such, ceramides can usually only be
modified by enzymes that exist in the membrane compartment that
the ceramide was produced in

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 Complex Sphingolipids
 The synthesis of complex sphingolipids occurs in the Golgi
apparatus.
 The bulky head groups of these sphingolipids make flipping
between membrane leaflets very unlikely without the aid of flip
passes. This means that without flip passes, sphingolipids are
restricted to the luminal Golgi leaflet/outer leaflet of the plasma
membrane.
 The two main type of complex sphingolipids are phospho- and
glycol-sphingolipids.

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 Phosphosphingolipids
 Phosphosphingolipids are linked by phosphodiester bonds.[5] In
mammals, the major phosphosphingolipids are gomyelins
(ceramide phosphocholines).
 In insects, it is ceramide phosphoethanolamines. The major
phosphosphingolipids in fungi are phyto ceramides
phosphoinositols and inositol phosphates.
 Aquatic organisms typically have sphingolipids where the
phosphate has been replaced with a phosphono- or arsenate group.
.

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 Glycosphingolipids
Glycosphingolipids have one or more carbohydrate groups in a
glycosidic linkage (usually; ceramide phophonositols are an
exception).

There are four classifications of glycosphingolipids:

1.Neutral glycosphingolipids: 1 or more uncharged sugars (i.e.
glucose, galactose, N-acetylglucosamine, N-acetylgalacoseamine,
fucose).
2.Acid glycosphingolipids: ionized functional groups (i.e.
phosphate or sulfate) attached to neutral or charged sugars.
3.Basic glycosphingolipids.
4.Amphoteric glycosphingolipids.

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 Cellular Signaling
Sphingolipids affect cell signaling by influencing the properties of
receptors directly. The exact pathway for signal transduction varies
depending on the exact function of the sphingolipids but there is a
general pathway.
Receptors are activated by cytokines (i.e. IL1, tumor necrosis
factor-ω), growth factors (i.e. platelet derived growth factor), or
other agonists.
Hydrolysis of membrane sphingolipid to ceramide is induced.
Sphingolipids either serve as a lipid second messenger, or can be
converted to downstream metabolites (i.e. sphingosine, sphingosine
1-phosphate, ceramide 1-phosphate).
Downstream targets (i.e. proteins kinases, phosphoprotein
phosphatases) that control cell behavior are either activated or
inhibited.

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 Sometimes, multiple sphingolipids within the same cell have
opposing signaling function. For example, ceraminde and
sphingosine 1-phosphate signal for conflicting functions of cell
growth. One signals for the inhibition of growth, while the other
signals for the stimulation of growth. (Or in other words, the
induction vs. inhibition of apoptosis.)
 The cellular pathway taken is determined by a
ceraminde/sphingosine 1-phosophate “rheostat” in deciding between
growth arrest/apoptosis and proliferation/survial.
Because sphingolipids are complex molecules, they can take diverse
input signals and produce multiple coordinated outputs. Signaling
from sphingolipids also alters membrane structure and behavior of
other membrane receptors and/or proteins.

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 5. Saccharolipids
 Saccharolipids describe compounds in which fatty acids are
linked directly to a sugar backbone, forming structures that
are compatible with membrane bilayers.
 In the saccharolipids, a monosaccharide substitutes for the
glycerol backbone present in glycerolipids and
glycerophospholipids.
The most familiar saccharolipids are the acylated
glucosamine precursors of the Lipid A component of the
lipopolysaccharides in Gram-negative bacteria.

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Typical lipid A molecules are disaccharides of glucosamine, which
are derivatized with as many as seven fatty-acyl chains. The minimal
lipopolysaccharide required for growth in Escherichia coli is Kdo2-
Lipid A, a hexa-acylated disaccharide of glucosamine (LipidA) that
is glycosylated with two 3-deoxy-D-manno-octulosonic acid (Kdo)
residues.

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 Chemical structure of lipid A as found in E. Coli

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 6. Polyketides
Polyketides are synthesized by polymerization of acetyl and
propionyl subunits by classic enzymes as well as interative and
multimodular enzymes that share mechanistic features with the
fatty acid synthesis.
They comprise a very large number of secondary metabolites
and natural products from animal, plant, bacterial, fungal and
marine sources, and have great structural diversity.
Many polyketides are cyclic molecules whose backbones are
often further modified by glycosylation, methylation,
hydroxylation, oxidation, and/or other processes.
Many commonly used anti-microbial, anti-parasitic, and anti-
cancer agents are polyketides or polyketide derivatives, such as
erythromycins, tetracylines, avermectins, and antitumor
epothilones.
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 7. Sterol lipids
Sterol lipids, such as cholesterol and its derivatives are an
important component of membrane lipids,along with the
glycerophospholipids and sphingomyelins.
The steroids, which also contain the same fused four-ring core
structure, have different biological roles as hormones and
signaling molecules.
The C18 steroids include the estrogen family whereas the C19
steroids comprise the androgens such as testosterone and
androsterone.
The C21 subclass includes the progestogens as well as the
glucocorticoids and mineralocorticoids. The secosteroids,
comprising various forms of vitamin D, are characterized by
cleavage of the B ring of the core structure.
Other examples of sterols are the bile acids and their
conjugates,which in mammals are oxidized derivatives of
cholesterol and are synthesized in the liver.
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Sterol is an organic compound with formula C17H28O, whose
molecule is derived from that of gonane by replacement of a
hydrogen atom in position 3 by a hydroxyl group. It is therefore an
alcohol of gonane. More generally, any compounds that contain the
gonane structure, additional functional groups, and/or modified ring
systems derived from gonane are called steroids. Therefore, sterols
are a subgroup of the steroids. They occur naturally in most
eukaryotes, including plants, animals, and fungi, and can also be
produced by some bacteria (however likely with different functions).

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 8. Prenol lipids
Prenol lipids are synthesized from the 5-carbon precursors
isopentenyl diphosphate and dimethylallyl diphosphate that are
produced mainly via the mevalonic acid (MVA) pathway.
The simple isoprenoids (linear alcohols, diphosphates, etc.) are
formed by the successive addition of C5 units, and are
classified according to number of these terpene units.
Structures containing greater than 40 carbons are known as
polyterpenes. Carotenoids are important simple isoprenoids
that function as anti-oxidants and as precursors of vitamin A.
Another biologically important class of molecules is
exemplified by the quinones and hydroquinones, which contain
an isoprenoid tail attached to a quinonoid core of non-
isoprenoid origin.
Vitamin E and vitamin K, as well as the ubiquinones, are
examples of this class.
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 Prokaryotes synthesize polyprenols (called bactoprenols) in which
the terminal isoprenoid unit attached to oxygen remains
unsaturated, whereas in animal polyprenols (dolichols) the
terminal isoprenoid is reduced.

Mevalonic Acid (MVA)

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 Prenol

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 Isopentenyl diphosphate

Dimethyl allyl diphosphate

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Signaling
In recent years, evidence has emerged showing that lipid signaling
is a vital part of the cell signaling.
Lipid signaling may occur via activation of GPCR's or nuclear
receptors, and members of several different lipid categories have
been identified as signaling molecules and cellular messengers.

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Signaling ……
These include sphingosine-1-phosphate, a sphingolipid
derived from ceramide that is a potent messenger molecule
involved in regulating calcium mobilization, cell growth,
apoptosis; diacylglycerol(DAG) and the
phosphatidylinositol phosphates (PIPs), involved in
calcium-mediated activation of protein kinase C; the
prostaglandins, which are one type of fatty-acid derived
eicosanoid involved in inflammation and immunity; the
steroid hormones such as estrogen, testosterone and
cortisol, which modulate a host of functions such as
reproduction, metabolism and blood pressure; and the
oxysterols such as 25-hydroxy-cholesterol that are Liver X
receptor (LXR) agonists.
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In addition to serving as a primary component of cellular
membranes and binding sites for intra- and intercellular
proteins, some glycerophospholipids in eukaryotic cells,
such as phosphatidylinositols and phosphatidic acids are
either precursors of, or are themselves, membrane-derived
second messengers.
Typically one or both of these hydroxyl groups are
acylated with long-chain fatty acids, but there are also
alkyl-linked and 1Z-alkenyl-linked (plasmalogen)
glycerophospholipids, as well as dialkylether variants in
prokaryotes.

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 Characterization of Physicochemical Properties

Solid Fat Content

The solid fat content (SFC) of a lipid influences many of its
sensory and physical properties, such as spreadability, firmness,
mouthfeel, processing and stability.
Food manufacturers often measure the variation of SFC with
temperature when characterizing lipids that are used in certain
foods, e.g., margarine and butter.
The solid fat content is defined as the percentage of the total
lipid that is solid at a particular temperature, i.e. SFC =
100Msolid/Mtotal, where Msolid is the mass of the lipid that is solid and
Mtotal is the total mass of the lipid in the food.
A variety of methods have been developed to measure the
temperature dependence of the solid fat content.
The density of solid fat is higher than the density of liquid oil,
and so there is an increase in density when a fat crystallizes and a
decrease
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 By measuring the density over a range of temperatures it is possible
to determine the solid fat content - temperature profile:

where r is the density of the lipid at a particular temperature,

and rL and rS are the densities of the lipid if it were completely
liquid or completely solid at the same temperature.
The density is usually measured by density bottles or dilatometry.
More recently, instrumental methods based on nuclear magnetic
resonance (NMR) have largely replaced density measurements,
because measurements are quicker and simpler to carry out
(although the instrumentation is considerably more expensive).

Desta S. 97 4/1/2023
 Basically, the sample is placed into an NMR instrument and a radio
frequency pulse is applied to it.
This induces a NMR signal in the sample, whose decay rate
depends on whether the lipid is solid or liquid.
The signal from the solid fat decays much more rapidly than the
signal from the liquid oil and therefore it is possible to distinguish
between these two components.
Techniques based on differential scanning calorimetry are also
commonly used to monitor changes in SFC.
These techniques measure the heat evolved or absorbed by a lipid
when it crystallizes or melts.
By making these measurements over a range of temperatures it is
possible to determine the melting point, the total amount of lipid
involved in the transition and the SFC-temperature profile.

Desta S. 98 4/1/2023
 Melting point
In many situations, it is not necessary to know the SFC over the
whole temperature range, instead, only information about the
temperature at which melting starts or ends is required.
A pure triacylglycerol has a single melting point that occurs at a
specific temperature.
Nevertheless, foods lipids contain a wide variety of different
triacylglycerols, each with their own unique melting point, and so
they melt over a wide range of temperatures.
Thus the "melting point" of a food lipid can be defined in a
number of different ways, each corresponding to a different amount
of solid fat remaining.
Some of the most commonly used "melting points" are:

Desta S. 99 4/1/2023
• Clear point. A small amount of fat is placed in a
capillary tube and heated at a controlled rate. The
temperature at which the fat completely melts and
becomes transparent is called the "clear point".
• Slip point. A small amount of fat is placed in a capillary
tube and heated at a controlled rate. The temperature at
which the fat just starts to move downwards due to its
weight is called the "slip point".
• Wiley melting point. A disc of fat is suspended in an
alcohol-water mixture of similar density and is then heated
at a controlled rate. The temperature at which the disc
changes shape to a sphere is called the "Wiley melting
point".
Desta S. 100 4/1/2023
 Cloud point
This gives a measure of the temperature at which
crystallization begins in a liquid oil.
A fat sample is heated to a temperature where all the crystals
are known to have melted (e.g., 130oC).
The sample is then cooled at a controlled rate and the
temperature at which the liquid just goes cloudy is determined.
This temperature is known as the cloud point, and is the
temperature where crystals begin to form and scatter light.
It is often of practical importance to have an oil which does
not crystallize when stored at 0oC for prolonged periods.
A simple test to determine the ability of lipids to withstand
cold temperatures without forming crystals, is to ascertain
whether or not a sample goes cloudy when stored for 5 hours at
0oC.

Desta S. 101 4/1/2023

 Smoke, Flash and Fire Points
These tests give a measure of the effect of heating on the
physicochemical properties of lipids.
They are particularly important for selecting lipids that
are going to be used at high temperatures, e.g. during
baking or frying.
The tests reflect the amount of volatile organic material in
oils and fats such as free fatty acids.
The smoke point is the temperature at which the sample
begins to smoke when tested under specified conditions.
A fat is poured into a metal container and heated at a
controlled rate in an oven.
The smoke point is the temperature at which a thin
continuous stream of bluish smoke is first observed.
Desta S. 102 4/1/2023
 The flash point is the temperature at which a flash
appears at any point on the surface of the sample due to the
ignition of volatile gaseous products.
The fat is poured into a metal container and heated at a
controlled rate, with a flame being passed over the surface
of the sample at regular intervals.
The fire point is the temperature at which evolution of
volatiles due to the thermal decomposition of the lipids
proceeds so quickly that continuous combustion occurs (a
fire).
Desta S. 103 4/1/2023
 Rheology
The rheology of lipids is important in many food applications.
Rheology is the science concerned with the deformation and flow
of matter.
Most rheological tests involve applying a force to a material and
measuring its flow or change in shape.
Many of the textural properties that people perceive when they
consume foods are largely rheological in nature, e.g., creaminess,
juiciness, smoothness, brittleness, tenderness, hardness, etc.
The stability and appearance of foods often depends on the
rheological characteristics of their components.

Desta S. 104 4/1/2023

 The flow of foods through pipes or the ease at which
they can be packed into containers are also determined by
their rheology.
Liquid oils are usually characterized in terms of their
flow properties (viscosity), whereas viscoelastic or plastic
"solids" are characterized in terms of both their elastic
(elastic modulus) and flow properties.
A wide variety of experimental techniques are available
to characterize the rheological properties of food
materials.
Desta S. 105 4/1/2023
 One of the most important rheological characteristics of
lipids is their "plasticity", because this determines their
"spreadability".
The plasticity of a lipid is due to the fact that fat crystals
can form a three-dimensional network that gives the
product some solid-like characteristics.
Below a certain stress (known as the "yield stress") the
product behaves like a solid with an elastic modulus
because the crystal network is not disrupted, but above this
stress it flows like a liquid because the crystal network is
continually disrupted.
Rheological techniques are therefore needed to measure
the change in deformation of a lipid when stresses are
applied.
Desta S. 106 4/1/2023
 Important chemical reactions of lipids
•Hydrolysis
•Rancidity
Hydrolysis •Hydrogenation
•Drying
•Acrolein formation
When fats are hydrolyzed with alkali or enzyme
Lipase the yield fatty acid and glycerol .
Lipases bring about splitting of fats in steps ,from
triglycerides to diglycerides to mono-glycerides and
finally to glycerol and fatty acids . Lipases may work in
the temperature range of 0° to 40° C .

Desta S. 107 4/1/2023

 The fats are hydrolyzed with alkali yielding the free fatty
acids which react with alkali to form salts.
the salts are soaps and this process is termed as
saponification .

Desta S. 108 4/1/2023

Oxidation ( Rancidity ) :-
Oxidation of fats in the air takes place along with
hydrolysis.
Oxidation takes place of double bonds of the fatty acids
, yielding short chain acids ; aldehydes etc.
this along with the fatty acids formed during hydrolysis
imparts a less palatable taste and odor to the fat .
It is termed as Rancidity .

Desta S. 109 4/1/2023

 Hydrogenation :-
By catalyst hydrogenation , unsaturated plant fats get
converted in to more saturated and solid fat .
It is generally done over finely divided nickel.
In the production of margarine and vegetable
shortening, this property has been exploited
commercially .

Desta S. 110 4/1/2023

 Hydrogenolysis :-
If excess of hydrogen , under pressure , is passed
through oil or fat in the presence of copper- chromium
catalyst, it split up into glycerol and higher aliphatic
alcohols.
The process of splitting a compound by means of
hydrogen is termed as hydrogenolysis.

Desta S. 111 4/1/2023

Acroline formation :-
If fats are heated in cooking ,some of the glycerol
released by the hydrolysis of the fat undergoes
decomposition to yield acroline which may be responsible
for head ache or severe smarting of the eye .

Desta S. 112 4/1/2023

 Determination of Total Lipid Concentration
Solvent Extraction
The fact that lipids are soluble in organic solvents, but
insoluble in water, provides the food analyst with a
convenient method of separating the lipid components in
foods from water soluble components, such as proteins,
carbohydrates and minerals.
In fact, solvent extraction techniques are one of the most
commonly used methods of isolating lipids from foods and
of determining the total lipid content of foods.

Desta S. 113 4/1/2023

 Semi-Continuous Solvent Extraction

Semi-continuous solvent extraction methods are commonly used to

increase the efficiency of lipid extraction from foods.
The Soxhlet method is the most commonly used example of a semi-
continuous method.
In the Soxhlet method a sample is dried, ground into small particles
and placed in a porous thimble.
The thimble is placed in an extraction chamber, which is suspended
above a flask containing the solvent and below a condenser.
The flask is heated and the solvent evaporates and moves up into the
condenser where it is converted into a liquid that trickles into the
extraction chamber containing the sample.
Eventually, the solvent builds up in the extraction chamber and
completely surrounds the sample.

Desta S. 114 4/1/2023

 The extraction chamber is designed so that when the solvent
surrounding the sample exceeds a certain level it overflows and
trickles back down into the boiling flask.
As the solvent passes through the sample it extracts the lipids and
carries them into the flask.
The lipids then remain in the flask because of their low volatility.
At the end of the extraction process, which typically lasts a few
hours, the flask containing the solvent and lipid is removed, the
solvent is evaporated and the mass of lipid remaining is measured
(Mlipid).
The percentage of lipid in the initial sample (Msample) can then be
calculated: %Lipid = 100 ´ (Mlipid/Msample). A number of instrument
manufacturers have designed modified versions of the Soxhlet
method that can be used to determine the total lipid content more
easily and rapidly (e.g. Soxtec).

Desta S. 115 4/1/2023

 Continuous Solvent Extraction

The Goldfish method is similar to the Soxhlet method

except that the extraction chamber is designed so that the
solvent just trickles through the sample rather than
building up around it.
This reduces the amount of time required to carry out
the extraction, but it has the disadvantage that channeling
of the solvent can occur, i.e., the solvent may preferentially
take certain routes through the sample and therefore the
extraction is inefficient.
This is not a problem in the Soxhlet method because the
sample is always surrounded by solvent.
Desta S. 116 4/1/2023
 Nonsolvent Liquid Extraction Methods.

A number of liquid extraction methods do not relay on

organic solvents, but use other chemicals to separate the
lipids from the rest of the food.
The Babcock, Gerber and Detergent methods are
examples of nonsolvent liquid extraction methods for
determining the lipid content of milk and some other dairy
products.

Desta S. 117 4/1/2023

 Babcock Method
A specified amount of milk is accurately pipetted into a specially
designed flask (the Babcock bottle).
Sulfuric acid is mixed with the milk, which digests the protein,
generates heat, and breaks down the fat globule membrane that
surrounds the droplets, thereby releasing the fat.
The sample is then centrifuged while it is hot (55-60oC) which
causes the liquid fat to rise into the neck of the Babcock bottle.
The neck is graduated to give the amount of milk fat present in
wt%.
The Babcock method takes about 45 minutes to carry out, and is
precise to within 0.1%.
It does not determine phospholipids in milk, because they are
located in the aqueous phase or at the boundary between the lipid and
aqueous phases.

Desta S. 118 4/1/2023

 Gerber Method
This method is similar to the Babcock method except that a
mixture of sulfuric acid and isoamyl alcohol, and a slightly
different shaped bottle, are used.
It is faster and simpler to carry out than the Babcock method.
The isoamyl alcohol is used to prevent charring of the sugars by
heat and sulfuric acid which can be a problem in the Babcock
method since it makes it difficult to read the fat content from the
graduated flask.
This method is used mainly in Europe, whilst the Babcock
method is used mainly in the USA.
As with the Babcock method, it does not determine
phospholipids.

Desta S. 119 4/1/2023

 Detergent Method
This method was developed to overcome the
inconvenience and safety concerns associated with
the use of highly corrosive acids.
A sample is mixed with a combination of
surfactants in a Babcock bottle.
The surfactants displace the fat globule
membrane which surrounds the emulsion droplets
in milk and causes them to coalesce and separate.
The sample is centrifuged which allows the fat to
move into the graduated neck of the bottle, where
its concentration can then be determined.
Desta S. 120 4/1/2023
 Chemical Techniques
Iodine Value
The iodine value of a substance is the weight of iodine
absorbed by 100 parts by weight of the substance when
determined by the following method.
Iodine bromide method (Ph. Eur. method 2.5.4)
Unless otherwise specified in the individual monograph, use the
following quantity of the substance being examined:
presumed iodine value less than 20 1.0 g
presumed iodine value 20 to 60 0.25 to 0.5 g
presumed iodine value 60 to 100 0.15 to 0.25 g
presumed iodine value more than 100 0.10 to 0.15 g.

Desta S. 121 4/1/2023

 Dissolve the specified quantity in 15 ml of chloroform in
an iodine flask fitted with a ground-glass stopper which is
dry or has been rinsed with glacial acetic acid, unless
otherwise specified in the monograph.
Add slowly 25 ml of iodine bromide solution, insert the
stopper and allow to stand in the dark for 30 minutes,
unless otherwise specified in the monograph, shaking
frequently.

Desta S. 122 4/1/2023

 Add 10 ml of a 10% w/v solution of potassium iodide
and 100 ml of water and titrate with 0.1M sodium
thiosulphate VS shaking vigorously until the yellow colour
almost disappears.
Add 5 ml of starch solution and complete the titration
adding the sodium thiosulphate solution dropwise.
Carry out the operation in exactly the same manner, but
without the substance being examined.

Calculate the iodine value from the expression

1.269v/w where v is the difference, in ml, between the
titrations and w is the weight, in g, of substance
taken.

Desta S. 123 4/1/2023

 The saponification value

The saponification value is the number of mg of

potassium hydroxide required to neutralise the free acids
and to saponify the esters in 1 g of the substance.

Dissolve 35 to 40 g of potassium hydroxide in 20 ml of

water and add sufficient ethanol (96%) to produce 1000
ml.
 Allow to stand overnight and pour off the clear liquid.

Desta S. 124 4/1/2023

Weigh 2 g of the substance into a 200-ml flask, add 25 ml
of the ethanolic solution of potassium hydroxide and boil
under a reflux condenser for 1 hour, rotating the contents
frequently.
While the solution is still hot, titrate the excess of alkali
with 0.5M hydrochloric acid VS using 1 ml of
phenolphthalein solution R1 as indicator.
Repeat the operation without the substance being
examined.
Calculate the saponification value from the expression
28.05v/w where v is the difference, in ml, between the
titrations and w is the weight, in g, of substance taken.

Desta S. 125 4/1/2023

 Acid value

The acid value is the number of mg of

potassium hydroxide required to neutralise the

free acid in 1 g of the substance when determined

by the following method, unless otherwise

specified in the monograph.

Desta S. 126 4/1/2023

 Unless otherwise specified in the monograph weigh 10
g of the substance being examined and add 50 ml of a
mixture of equal volumes of ethanol (96%) and ether
that has been neutralised with 0.1M potassium hydroxide
VS using 0.5 ml of phenolphthalein solution R1 as
indicator.
When the substance has completely dissolved, titrate
with 0.1M potassium hydroxide VS, shaking constantly
until a pink colour that persists for at least 15 seconds is
produced.
Calculate the acid value from the expression
5.610v/w, where v is the volume, in ml, of potassium
hydroxide solution required and w is the weight, in g, of
substance taken.
Desta S. 127 4/1/2023
 The unsaponifiable matter

The unsaponifiable matter is the percentage

content, w/w, of material not volatile at 100° to 105°
that is obtained by extraction with an organic
solvent from the saponified substance being
examined.
Use Method I unless otherwise specified in the
monograph. Use ungreased ground-glass glassware
for each method.

Desta S. 128 4/1/2023

 To 2.0 to 2.5 g of the substance being examined in a 250-
ml flask add 25 ml of 0.5M ethanolic potassium hydroxide
and boil under a reflux condenser in a water bath for 1 hour,
swirling the contents frequently.
Wash the contents of the flask into a separating funnel
with the aid of 50 ml of water and, while the liquid is still
slightly warm, extract by shaking vigorously with three 50-
ml quantities of peroxide-free ether, rinsing the flask with
the first quantity of ether.
Desta S. 129 4/1/2023
 Mix the ether solutions in a separating funnel
containing 20 ml of water. (If the ether solutions contain
solid suspended matter, filter them into the separating
funnel through a fat-free filter paper and wash the filter
paper with peroxide-free ether.)
Gently rotate the separating funnel for a few minutes
without violent shaking, allow the liquids to separate and
discard the aqueous layer.
Wash the ether solution by shaking vigorously with
two 20-ml quantities of water and then treat with three
20-ml quantities of 0.5M potassium hydroxide, shaking
vigorously on each occasion, each treatment being
followed by washing with 20 ml of water

Desta S. 130 4/1/2023

 Finally wash with successive 20-ml quantities of water until the
aqueous layer is no longer alkaline to phenolphthalein solution R1.
Transfer the ether extract to a weighed flask, rinsing the separating
funnel with peroxide-free ether, distil the ether and add 3 ml of
acetone to the flask.
With the aid of a gentle current of air, remove the solvent
completely from the flask, which is almost entirely immersed in
boiling water and preferably held obliquely and rotated.
Dry to constant weight at a temperature not exceeding 80° and
dissolve the contents of the flask in 10 ml of freshly boiled ethanol
(96%), previously neutralised to phenolphthalein solution R1.
Titrate with 0.1M ethanolic sodium hydroxide VS using
phenolphthalein solution R1 as indicator.

Desta S. 131 4/1/2023

 Calculate the unsaponifiable matter as a percentage of
the substance being examined.
If the volume of 0.1M ethanolic sodium hydroxide VS
required exceeds 0.1 ml, the amount of residue weighed
cannot be taken as the unsaponifiable matter and the test
must be repeated.

Desta S. 132 4/1/2023

 The ester value

The ester value is the number of mg of potassium

hydroxide required to saponify the esters present in 1 g of
the substance.
Determine the acid value, Appendix X B, of the
substance being examined and the saponification value.
Calculate the ester value by subtracting the acid value
from the saponification value.

Desta S. 133 4/1/2023

 The acetyl value
The acetyl value of a substance is the number of mg of
potassium hydroxide required to neutralise the acetic acid
liberated by the hydrolysis of 1 g of the acetylated
substance.
Determine the saponification value, Acetylate by the
following method.
 To 10 g in a 200-ml Kjeldahl flask add 20 ml of acetic
anhydride.
Support the flask on a sheet of heat resistant material in
which a hole about 4 cm in diameter has been cut and heat
with a small, naked flame, not more than 25 mm in height
and which does not impinge on the bottom of the flask.
Desta S. 134 4/1/2023
 Boil gently under a reflux air condenser for 2 hours,
allow to cool, pour into 600 ml of water contained in a
large beaker, add 0.2 g of pumice powder and boil for 30
minutes.
Cool, transfer to a separating funnel and discard the
lower layer.
Wash the acetylated product with three or more 50-ml
quantities of a warm, saturated solution of sodium chloride
until the washings are no longer acidic to litmus paper
Desta S. 135 4/1/2023
 Finally shake with 20 ml of warm water and remove
the aqueous layer as completely as possible.
 Pour the acetylated substance into a small dish, add
1 g of powdered anhydrous sodium sulphate, stir
thoroughly and filter through a dry, pleated filter paper.
Determine the saponification value of the acetylated
substance.
Calculate the acetyl value from the expression
1335(b-a)/(1335-a) where a is the saponification
value of the substance and b is the saponification
value of the acetylated substance.

Desta S. 136 4/1/2023

 •Reichert Miessel’s value
Reichert- Meissel’s Value: - It may be considered to be
a measure of volatile fatty acids present as glycerids in
oils and fats. Its defined as the number of a ml of 0.1N
Potassium Hydroxide solution required to neutralize the
distillate of 5g of hydrolyzed fats or oil.
It finds use for determining the purity of butter, ghee.
The R.M. Value for pure butter and gee lies in-between
20 – 30 , for coconut oil between 6 – 8.5 and for palm
kernel oil between 4 – 7 .

Desta S. 137 4/1/2023

 •Polneski’s value
Polenski’s Value: -
It may be defined as number of ml of 0.1N Potassium
hydroxide solution needed to neutralize the steam volatile,
but water insoluble fatty acids obtained from the distillate
of 5g of hydrolyzed oil or fat .
The water insoluble fatty acids have been obtained by
filtration of the distillate in R. M value of determination.
They are dissolved in alcohol and titrated against 0.1N
Potassium hydroxide.
Desta S. 138 4/1/2023
 Detergents
Soap, cleansing agent or detergent (see Detergents),
made from animal and vegetable fats, oils, and greases;
chemically, the sodium or potassium salt of a fatty acid,
formed by the interaction of fats and oils with alkali.
Oils and fats used are compounds of glycerin and a fatty
acid, such as palmitic, or stearic acid.
When these compounds are treated with an aqueous
solution of an alkali, such as sodium hydroxide—a
process called saponification—they decompose, forming
glycerin and the sodium salt of the fatty acid.

Desta S. 139 4/1/2023

The fat palmitin, for example, which is the ester of
glycerin and palmitic acid, yields sodium palmitate (soap)
and glycerin upon saponification.
The fatty acids required for soap making are supplied by
tallow, grease, fish oils, and vegetable oils such as coconut
oil, olive oil, palm oil, soybean oil, and corn oil.
Hard soaps are made from oils and fats that contain a
high percentage of saturated acids, which are saponified
with sodium hydroxide.
Desta S. 140 4/1/2023
Desta S. 141 4/1/2023