This document provides information on lipids including fatty acids, phospholipids, sphingolipids, cerebrosides, steroids, bile acids, prostaglandins, waxes, terpenes, lipoamino acids, lipoproteins, proteolipids, and lipopolysaccharides. It discusses their structures, properties, functions, classification, and quantitative and qualitative estimation methods. Specific lipid types like fatty acids, glycerophospholipids, sphingolipids, bile acids, sterols, lipoproteins, and proteolipids are described in further detail including their production, roles in disease, and catabolism.

1. LIPIDS
Unit III: Lipids: Definition, classification, structure, properties and function of fatty
acids, essential fatty acids, phospholipids, sphingolipids, cerebroside, steroids, bile
acids, prostaglandins, waxes, terpenes, lipoamino acids, lipoproteins, proteolipids,
lipopolysaccharides, iodine test, saponification, acid value, RM number, Brief account
of various quantitative and qualitative methods of estimation.
TEJASVI NAVADHITAMASTU
“Let our (the teacher and the taught) learning be radiant”
Let our efforts at learning be luminous and filled with joy, and
endowed with the force of purpose
Unit-3

3. FATTY ACIDS

8. TESTS TO CHECK PURITY OF FATS AND OILS

9. Clycerophospholipids

15. Catabolism
Degradation of glycosphingolipids occurs in the
lysosome, which contains digestive enzymes in
animal cells. The lysosome breaks down the
glycosphingolipid to its primary components, fatty
acids, sphingosine, and saccharide.
Role in disease
A defect in the degradation of glucocerebrosides
is Gaucher's disease. The corresponding defect for
galactocerebrosides is Krabbe disease

20. Bile acid
Bile acids are steroid acids found predominantly in
the bile of mammals and other vertebrates. Different
molecular forms of bile acids can be synthesized in
the liver by different species.[1][2][3] Bile acids are
conjugated with taurine or glycine in the liver, forming bile
salts
Primary bile acids are those synthesized by the
liver. Secondary bile acids result from bacterial actions in
the colon. In humans, taurocholic acid and glycocholic
acid(derivatives of cholic acid) and taurochenodeoxycholic
acid and glycochenodeoxycholic acid (derivatives
of chenodeoxycholic acid) are the major bile salts in bile
and are roughly equal in concentration.[4] The conjugated
salts of their 7-alpha-dehydroxylated
derivatives, deoxycholic acid and lithocholic acid, are also
found, with derivatives of cholic, chenodeoxycholic and
deoxycholic acids accounting for over 90% of human biliary
bile acids.[4]

22. Production
Bile acid synthesis occurs in liver cells which synthesize primary bile
acids (cholic acid and chenodeoxycholic acid in humans) via cytochrome
P450-mediated oxidation of cholesterol in a multi-step process.
Approximately 600 mg of bile salts are synthesized daily to replace
bile acids lost in the feces..
Prior to secreting any of the bile acids (primary or secondary, see below),
liver cells conjugate them with one of two amino acids, glycine or taurine, to
form a total of 8 possible conjugated bile acids. These conjugated bile
acids are often referred to as bile salts because of their physiologically-
important acid-base properties.
When these bile acids are secreted into the lumen of the intestine, bacterial
partial dehydroxylation and removal of the glycine and taurine groups forms
the secondary bile acids, deoxycholic acid and lithocholic acid. Cholic acid
is converted into deoxycholic acid and chenodeoxycholic acid into lithocholic
acid. All four of these bile acids can be taken back up into the blood stream,
return to the liver, and be re-secreted in a process known as enterohepatic
circulation.[2][3]

23. Functions
1. As amphipathic molecules with hydrophobic and hydrophilic regions, conjugated
bile salts sit at the lipid/water interface and, at the right concentration, form
micelles.[9] The added solubility of conjugated bile salts aids in their function by
preventing passive re-absorption in the small intestine.
2. Bile acid-containing micelles aid lipases to digest lipids and bring them near the
intestinal brush border membrane, which results in fat absorption.[5]
3. Synthesis of bile acids is a major route of cholesterol metabolism in most species
other than humans. The body produces about 800 mg of cholesterol per day and
about half of that is used for bile acid synthesis producing 400–600 mg daily.
4. Human adults secrete between 12-18 g of bile acids into the intestine each day,
mostly after meals. The bile acid pool size is between 4–6 g, which means that bile
acids are recycled several times each day. About 95% of bile acids are reabsorbed
by active transport in the ileum and recycled back to the liver for further secretion
into the biliary system and gallbladder.
5. Bile acids have other functions, including eliminating cholesterol from the body,
driving the flow of bile to eliminate certain catabolites (including bilirubin),
emulsifying fat-soluble vitamins to enable their absorption, and aiding in motility
and the reduction of the bacteria flora found in the small intestine and biliary
tract.[4]

24. Lipoproteins
Lipoproteins are molecules made of
proteins and fat. They carry
cholesterol and similar substances
through the blood. A blood test can
be done to measure a specific type
oflipoprotein called lipoprotein-a,
or Lp(a). A high level of Lp(a) is
considered a risk factor for heart
disease
A lipoprotein is a biochemical assembly that contains both proteins and lipids,
bound to the proteins, which allow fats to move through the water inside and
outside cells. The proteins serve to emulsify the lipid molecules.
Manyenzymes, transporters, structural proteins, antigens, adhesins,
and toxins are lipoproteins. Examples include theplasma lipoprotein
particles classified under high-density (HDL) and low-density (LDL) lipoproteins,
which enable fats to be carried in the blood stream, the transmembrane
proteins of the mitochondrion and the chloroplast, and bacterial lipoproteins.[1

25. Classification
Lipoproteins may be classified as follows, listed from larger and less dense to
smaller and denser. Lipoproteins are larger and less dense when the fat to protein
ratio is increased. They are classified on the basis
of electrophoresis and ultracentrifugation.
•Chylomicrons carry triglycerides (fat) from the intestines to the liver, to skeletal
muscle, and to adipose tissue.
•Very-low-density lipoproteins (VLDL) carry (newly synthesised) triglycerides from
the liver to adipose tissue.
•Intermediate-density lipoproteins (IDL) are intermediate between VLDL and LDL.
They are not usually detectable in the blood when fasting.
•Low-density lipoproteins (LDL) carry 3,000 to 6,000 fat molecules (phospholipids,
cholesterol, triglycerides, etc.) around the body. LDL particles are sometimes
referred to as "bad" lipoprotein because concentrations, dose related, correlate
with atherosclerosis progression.
• large buoyant LDL (lb LDL) particles
• small dense LDL (sd LDL) particles
• Lipoprotein(a) is a lipoprotein particle of a certain phenotype
•High-density lipoproteins (HDL) collect fat molecules (phospholipids, cholesterol,
triglycerides, etc.) from the body's cells/tissues, and take it back to the liver. HDLs
are sometimes referred to as "good" lipoprotein because higher concentrations
correlate with low rates of atherosclerosis progression and/or regression.

26. Density (g/
mL)
Class
Diameter
(nm)
% protein % Chol
%
phospholipi
d
% TAG
&
cholesterol
ester
>1.063 HDL 5–15 33 30 29 4
1.019–
1.063
LDL 18–28 25 50 21 8
1.006–
1.019
IDL 25–50 18 29 22 31
0.95–1.006 VLDL 30–80 10 22 18 50
<0.95
Chylomicro
ns
100-1000 <2 8 7 84
For Young Healthy Research Subjects, ~70 Kg, 154 Lb, The Following Applies:

27. Function
The handling of lipoprotein particles
in the body is referred to
as lipoprotein particle metabolism. It
is divided into two
pathways, exogenous and endogeno
us, depending in large part on
whether the lipoprotein particles in
question are composed chiefly of
dietary (exogenous) lipids or whether
they originated in the liver
(endogenous), through de novo
synthesis of triacylglycerols.
The hepatocytes are the main
platform for the handling of
triacylglyerols and cholesterol; the
liver can also store certain amounts
of glycogen and triacylglycerols.
While adipocytes are the main
storage cells for triacylglycerols, they
do not produce any lipoproteins.

34. PROTEOLIPIDS
Proteolipids can be defined as all proteins containing
covalently bound lipid moieties, including fatty acids,
isoprenoids, cholesterol and glycosylphosphatidylinositol

35. During the course of the study of brain sulfatides with Lees MB, Folch J described
for the first time in 1951 the presence of special proteins in rat brain myelin which
could be solubilized in organic solvents (chloroform / methanol / water mixtures)
(Folch J et al., J Biol Chem 1951, 191, 807). These substances were named
"proteolipides" and were considered as a novel lipoprotein but quite different from
the other known lipoproteins.
These proteolipids were shown to be present mainly in neural tissues but also
in heart, kidney, liver, and muscles but absent from blood plasma.
During thirty years the definition of proteolipids was exclusively used to refer to
a family of various proteins which are related by their solubility in mixtures of
chloroform and methanol (Lees MB et al., Biochim Biophys Acta 1979, 559,
209). Thus, the archetypal proteolipid found initially in myelin is now known as
"proteolipid protein" or PLP.
The presence of fatty acids covalently associated with hydrophobic proteins
was first described in Gram-negative bacteria but rapidly extended to myelin
PLP and to the Ca++-dependent ATPase complex of sarcoplasmic reticulum.
These discoveries led to the new definition for proteolipid : a protein that
contains a lipid moiety as part of its primary structure.

36. Curiously, only two types of acylated proteins have been identified :
- Myristoylated proteins
Myristic acid (C14:0) is bound to the amino-terminal glycine residue
(stable amide linkage)
- Palmitoylated proteins
Palmitic acid (C16:0) is bound to side chains of cystein residues
(labile thioester linkage). Other fatty acids can also be present
(C16:1, C18:2, C20:0 ..)

37. MYRISTOYLATED PROTEINS
The first proteins to be demonstrated to contain myristic acid were calcineurin B
(Aitken A et al., FEBS Lett 1982, 150, 314) and the catalytic subunit of the cyclic AMP-
dependent protein kinase (Carr SA et al., Proc Natl Acad Sci USA 1982, 79, 6128).
It was shown that myristic acid (R2) was attached through an amide linkage to the a-
amino group of glycine (R1) at the N-terminus of both proteins :
R1--NH--CO--R2
Later, a wide range of proteins of viral and cellular origin have been shown to be
modified by acylation with myristic acid (Olson EN, Prog Lipid Res 1988, 27, 177).
Myristoylated proteins are localized to the cytosol or to cellular membranes and
sometimes to both. Membrane-bound myristoylated proteins interact tightly with the
bilayer so that drastic conditions may be used to release them from membranes (Olson
EN et al., J Biol Chem 1986, 261, 2458). It is now well established that myristoylation is
able to direct soluble proteins to membranes but the specificity of targeting remains
unclear.
The function for myristoylation is also not well known. It was speculated that these
proteins may represent enzymes involved in lipid metabolism or carrier proteins.

38. Wax
Waxes are a class of chemical compounds that are malleable near ambient
temperatures. They are also a type of lipid. Characteristically, they melt above 45 °C
(113 °F) to give a low viscosity liquid. Waxes are insoluble in water but soluble in
organic, nonpolar solvents. All waxes are organic compounds, both synthetically and
naturally occurring.
Plant and animal waxes
Waxes are synthesized by many plants and animals. Those of animal origin typically
consist of wax esters derived from a variety of carboxylic acids and fatty alcohols. In
waxes of plant origin characteristic mixtures of unesterified hydrocarbons may
predominate over esters.[1] The composition depends not only on species, but also on
geographic location of the organism. Because they are mixtures, naturally produced
waxes are softer and melt at lower temperatures than the pure components.[citation needed]
Chemical Structure[edit]
Wax is a type of long chain apolar lipid which made up of various n-alkanes, ketones,
primary alcohol, secondary alcohols, monoesters, beta diketones, aldehydes,etc.
Waxes will form protective coating on plants and fruits, and in animal (example:
beewax, whale spermaceti, etc.).
More commonly, wax is ester of alcohol and fatty acids. They differ from fats since
they don’t have triglyceride ester of three fatty acids. Waxes are water resistant, so
they are insoluble in water

39. Animal waxes[edit]
The most commonly known animal wax is beeswax, but other insects secrete
waxes. A major component of the beeswax used in constructing honeycombs is
the ester myricyl palmitate which is an ester of triacontanol and palmitic acid. Its
melting point is 62-65 °C.Spermaceti occurs in large amounts in the head oil of
the sperm whale. One of its main constituents is cetyl palmitate, another ester of
a fatty acid and a fatty alcohol. Lanolin is a wax obtained from wool, consisting of
esters of sterols.[2]
Plant waxes[edit]
Plants secrete waxes into and on the surface of their cuticles as a way to control
evaporation, wettability and hydration.[3] The epicuticular waxes of plants are mixtures
of substituted long-chain aliphatic hydrocarbons, containing alkanes, alkyl esters, fatty
acids, primary and secondary alcohols, diols, ketones,aldehydes.[1] From the
commercial perspective, the most important plant wax is Carnauba wax, a hard wax
obtained from the Brazilian palm Copernicia prunifera. Containing the ester myricyl
cerotate, it has many applications, such as confectionery and other food coatings, car
and furniture polish, floss coating,surfboard wax, and other uses. Other more
specialized vegetable waxes include candelilla wax and ouricury wax.
One component of beeswax is myricin (myricyl palmitate, CH3(CH2)14COO(CH2)12CH3).
Myricyl palmitate is a saturated 16 carbon fatty acid esterified to a 30 carbon alcohol.

40. Properties[edit]
Due to the versatility of waxes, nature has manipulated them for their water-resistant properties,
colligative properties (high melting point, relatively low viscosity at high temperatures, transparency,
etc.) and coating properties.
Types of Waxes[edit]
1. Beeswax – for consumption
2. Chinese Wax – for polishes
3. Ear Wax – used as a protective layer over the ear membrane
4. Lanolin – for rust prevention and cosmetics
5. Shellac – used as a wood sealant
6. Spermaceti – for cosmetics and leatherworking
7. Vegetable (many different types extracted from plants) – used as a protective layer on the plant
to prevent loss of water
8. Mineral – used as fine polishes
9. Petroleum – fuels, paints, culinary, candles
10.Synthetic – modified waxes for use in the medical field
Functions and Applications[edit]
Waxes contain many functions in society. Man has manipulated and synthesized many waxes to be
used for cosmetics, sealants and lubricants, insecticides, UV protection, energy reserves, food, etc.

41. LIPOAMINO ACIDS
Several classes of complex lipids devoid of phosphorus have one amino
acid linked to both a long-chain alcohol and a fatty acid or to a glycerolipid,
they are sometimes named lipoamino acids.
Simple forms of these lipoamino acids containing only amino acid and fatty
acid(s) are described in the "simple lipids" part.
They are present exclusively in Bacteria and lower plants (fern, algae,
protozoa)
Two groups of complex lipoamino acids are known:
1 - Lipids having an amino acid with N-acyl and/or ester linkages
2 - Lipids having a glycerol and an amino acid with ether linkage

42. LIPOAMINO ACIDS :
N-ACYL and ESTER DERIVATIVES of AMINO
ACIDS
Several types of derivatives are known
according to their amino acid moiety:
1 - Lysine-containing lipids
Some of them are known as Siolipin A. In these
compounds lysine is N-linked to a fatty acid
(normal or hydroxylated, R1) and to a fatty
alcohol (R2) (ester link). They are found
in Streptomyces species of bacteria.

43. 2 - Ornithine-containing lipids
In these lipids ornithine is linked to a fatty acid (R1) by an
amide link and to a long-chain fatty alcohol (R2) by an
ester link.
The fatty acid chain (R1) has 16 to 18 carbon atoms and
the fatty alcohol may have a cyclopropane ring. They
occur in photosynthetic purple bacteria (Gorshein A,
Biochim Biophys Acta 1968, 152, 358).
Less complex forms containing ornithine linked to fatty
acids only were also described.
Other ornithine-containing lipids are found in Gram
negative bacteria and have been reported in some Gram-
positives, like Mycobacterium and Streptomyces species
but are absent in Archaea and Eukarya (Geiger O et al.,
Prog Lipid Res 2010, 49, 46). They are commonly formed
of a 3-hydroxy fatty acyl group that is attached in amide
linkage to the a-amino group of ornithine an a second
fatty acyl group is ester-linked to the 3-hydroxy position of
the first fatty acid.

44. 3- Glycine-containing lipids
The glycine-containing lipids have been identified in the gliding
bacterium Cytophaga johnsonae (Kawazoe R et al., J Bacteriol
199, 173, 5470) and the Gram-negative sea-water
bacterium Cyclobacterium marinus (Batrakov SG et al., Chem
Phys Lipids 1999, 99, 139). These lipids consist of the amino acid
glycine and two fatty acyl residues, using the acyl-oxyacyl
structure. The structure of glycine lipid from C. marinus is mainly
a N-[3-D-(13-methyltetradecanoyloxy)- 15-
methylhexadecanoyl]glycine (see figure below). In this structure,
an iso-3-hydroxyfatty acyl group is amide-linked to glycine and its
3-hydroxy group is esterified to another iso-fatty acid.

45. LIPOAMINO ACIDS WITH ETHER LINKAGE
These lipids are glycerolipids and are mainly derived from homoserine, they are characterics of algae.
Some forms have an alanine moiety instead of homoserine. As the polar head may be considered derived
from betaine (N,N,N-trimethyl glycine), these lipids are commonly named betain lipids.
The homoserine-derived lipids were
first identified in a yellow-green
algae, Ochromonas
danica (Chrysophyceae) where they
account for more than 50% of total
lipids(Brown AE et al., Biochemistry
1974, 13, 3476). It has been
suggested that homoserine-derived
lipids, which are formed by an ether
linkage between homoserine and a
diacylglycerol molecule, are widely
distributed and even found in some
higher plants (Rozentsvet OA et al.,
Phytochemistry 2000, 54, 401).

46. Alanine-derived lipids (diacylglyceryl hydroxymethyltrimethyl-b-alanine) was first identified
in Ochromonas danica (Vogel G et al., Chem Phys lipids 1990, 52, 99) and was shown to
replace the homoserine-derived lipids in brown algae but are absent in the greens
(Eichenberger W, Plant Physiol Biochem 1993, 31, 213).
Another betain lipid, diacylglyceryl carboxyhydroxymethylcholine, was then discovered
in Pavlova lutheri (Haptophyceae) (Kato M et al., Phytochemistry 1994, 37, 279).
Lysine-containing diacylglycerol was isolated from Mycobacterium phlei strain IST (Lerouge P
et al., Chem Phys Lipids 1988, 49, 161). Lysine is esterified to 1,2- diglyceride via an ester
linkage and the major fatty acyl substitutions are palmitic and tuberculostearic acid.

47. PALMITOYLATED PROTEINS
These proteins are the most extensively studied among proteolipids and the first member among them to be
identified was the myelin PLP which represents the major component of the myelin proteins (at least 40%).
The long-chain fatty acids (R2, mainly C16:0, C18:0 and C18:1) constitute about 2-4% of the PLP dry weight
and are covalently bound by thioester linkages to cystein residues (R1).
R1--S--CO--R2
The presence of thioester bonds was demonstrated by in vitro and in vivo acylation (Ross NW et al., J
Neurosci Res 1988, 21, 35; Bizzozero OA et al., J Neurochem 1990, 55, 1986).
PLP was shown to be palmitoylated with acyl-CoA by a non-enzymatic mechanism and depalmitoylated by
a specific myelin-associated acyltransferase.
The extreme hydrophobicity of PLP is easily explained by a composition of about 50% apolar amino acid
residues and a high degree of fatty acid acylation (Weimbs T et al., Biochemistry 1992, 31, 12289).
Besides myelin PLP, several other membrane proteins were shown to be S-palmitoylated. The best known
examples are the followings :
- myelin P0 glycoprotein in peripheral nervous system (Bizzozero OA et al., Anal Biochem 1989, 180, 59).
- ligatin in neonatal enterocytes (Jakoi ER et al., J Biol Chem 1987, 262, 1300).
- lung surfactant proteolipid (Stults JT et al., Am J Physiol 1991, 261, L118).
- rhodopsin in retina cells (O'Brien P et al., J Biol Chem 1987, 262, 5210).
- sodium channel polypeptide (Levinson SR et al., Biophys J 1986, 49, 378A).
- P-selectin in vascular endothelium (Fujimoto T et al., J Biol Chem 1993, 268, 11394).
- band 3 protein in erythrocytes (Okudo K et al. J Biol Chem 1991, 266, 16420).
- hepatic asialoglycoprotein receptor (Zeng FY et al., J Biol Chem 1995, 270, 21382).
- glycoprotein proteolipids from Sindbis virus (Schmidt MFG et al., Proc Natl Acad Sci USA 1979, 76,
1687).

48. More than 20 proteins modified by covalent palmitic acid were reviewed in
1988 (Olson EN, Prog Lipid Res 1988, 27, 177) and 14 were added in 1994
(Bizzozero OA et al., Neurochem Res 1994, 19, 923).
A phylogenetic conservation of fatty acid acylation was demonstrated in
studying brain myelin from amphibians, reptiles, birds and mammals,
suggesting a critical role of this post-translational modification for PLP function
(Bizzozero OA et al., Neurochem Res 1999, 24, 269). In all species, PLP
contains about 3% (w/w) of bound fatty acids, 78% of them being C16:0,
C16:1, C18:0 and C18:1. Curiously, hydroxy and branched-chain fatty acids
are absent. While discrepancies are found concerning the fatty acid to protein
stoichiometry, it is now accepted that no more than 3 moles of fatty acids are
bound to one mole of PLP (MW = 25000). Interestingly, PLP appears to be
strongly associated in situ with acidic phospholipids, mostly
phosphatidylserine. It is estimated that about 15 molecules of phospholipids
form a boundary lipid matrix around a molecule of PLP.

49. Terpene
Terpenes (/ˈtɜːrpiːn/) are a large and diverse class of organic compounds,
produced by a variety of plants, particularly conifers, and by some insects.[1][2]
They often have a strong odor and may protect the plants that produce them by
deterring herbivores and by attracting predators and parasites of herbivores.[3][4]
Although sometimes used interchangeably with
"terpenes", terpenoids (or isoprenoids) are modified terpenes as they contain
additional functional groups, usually oxygen-containing.[5]
Terpenes are hydrocarbons.
Terpenes are also major biosynthetic building blocks. Steroids, for example,
are derivatives of the triterpene squalene.
Terpenes and terpenoids are the primary constituents of the essential oils of
many types of medicinal plants and flowers. Essential oils are used widely as
fragrances in perfumery, and in medicine and alternative medicines such
as aromatherapy. Synthetic variations and derivatives of natural terpenes and
terpenoids also greatly expand the variety of aromas used in perfumery and
flavors used in food additives. Vitamin A is a terpenoid.

50. Structure and biosynthesis
Isoprene phase
Mevalonic acid pathway
MEP/DOXP pathway
Isoprenoid
The five-carbon unit that constitutes the basic building block of isoprenoids is
a hydrocarbon called isoprene. Isoprene (2-methyl-1,3-butadiene) is a
branched-chain unsaturated hydrocarbon, unsaturated meaning it contains
one or more double bonds between carbon atoms.

51. Lipopolysaccharide
Lipopolysaccharides (LPS), also known as lipoglycans
and endotoxins, are large molecules consisting of
a lipid and apolysaccharide composed of O-antigen,
outer core and inner core joined by a covalent bond; they
are found in the outer membrane of Gram-negative
bacteria, and elicit strong immune responses in animals.
The term lipooligosaccharide ("LOS") is used to refer to a
low-molecular-weight form of bacterial lipopolysaccharides.

53. Functions in bacteria
LPS is the major component of the outer membrane of Gram-
negative bacteria, contributing greatly to the structural integrity of the bacteria,
and protecting the membrane from certain kinds of chemical attack. LPS also
increases the negative charge of the cell membrane and helps stabilize the
overall membrane structure. It is of crucial importance to gram-negative
bacteria, whose death results if it is mutated or removed. LPS induces a
strong response from normal animal immune systems. It has also been
implicated in non-pathogenic aspects of bacterial ecology, including surface
adhesion, bacteriophage sensitivity, and interactions with predators such
as amoebae.
LPS is required for the proper conformation of Omptin activity; however,
smooth LPS will sterically hinder omptins.

54. Composition
It comprises three parts:
1.O antigen (or O polysaccharide)
2.Core oligosaccharide
3.Lipid A
The saccharolipid Kdo2-Lipid A.
Glucosamine residues in blue, Kdo
residues in red, acyl chains in black and
phosphate groups in green.

55. O-antigen[edit]
A repetitive glycan polymer contained within an LPS is referred to as the O antigen, O polysaccharide,
or O side-chain of the bacteria. The O antigen is attached to the core oligosaccharide, and comprises
the outermost domain of the LPS molecule. The composition of the O chain varies from strain to strain.
For example, there are over 160 different O antigen structures produced by different E.
coli strains.[4] The presence or absence of O chains determines whether the LPS is considered rough or
smooth. Full-length O-chains would render the LPS smooth, whereas the absence or reduction of O-
chains would make the LPS rough.[5] Bacteria with rough LPS usually have more penetrable cell
membranes to hydrophobic antibiotics, since a rough LPS is more hydrophobic.[6] O antigen is exposed
on the very outer surface of the bacterial cell, and, as a consequence, is a target for recognition by
host antibodies.
Core[edit]
Main article: Core oligosaccharide
The Core domain always contains an oligosaccharide component that attaches directly to lipid A and
commonly contains sugars such as heptose and 3-deoxy-D-mannooctulosonic Acid (also known as
KDO, keto-deoxyoctulosonate).[7] The LPS Cores of many bacteria also contain non-carbohydrate
components, such as phosphate, amino acids, and ethanolamine substituents.
Lipid A[edit]
Main article: Lipid A
Lipid A is, in normal circumstances, a phosphorylated glucosamine disaccharide decorated with
multiple fatty acids. These hydrophobic fatty acid chains anchor the LPS into the bacterial membrane,
and the rest of the LPS projects from the cell surface. The lipid A domain is responsible for much of the
toxicity of Gram-negative bacteria. When bacterial cells are lysed by the immune system, fragments of
membrane containing lipid A are released into the circulation, causing fever, diarrhea, and possible fatal
endotoxic shock (also called septic shock). The Lipid A moiety is a very conserved component of the
LPS.[8]

56. ESTIMATION OF CHOLESTEROL BY ZAKS METHOD
PRINCIPLE :
Cholesterol is a steroid lipid, amphipathic in nature. It consistes of basic
cyclopentano perhydro phenothrene nucleus. It is synthesized in liver from
Acetyle CoA. It acts as a precursor for steroid hormones and vitamin D. The
serum cholesterol exists in 2 forms.
Esterified form and Free Form
The proteins present in the serum sample are first precipitated by adding Fecl3-
CH3 COOH reagent. The protein free filterate is treated with conc. H2SO4. In
the presence of conc. H2SO4, cholesterol present in the serum gets dehydrated
to form cholesterol 3, 5 diene in presence of excess H2SO4 and by the catalytic
action of Fe3+ ions a red coloured complex is formed. The intensity of red colour
is measured at 560 nm.

57. Reagents :
1. FeCl3-CH3COOH reagent(0.05%)–0.05gms of FeCl3 is
dissolved in 100ml of aldehyde free CH3COOH.
2. conc. H2SO4
3. Cholesterol standard
4. Stock Solution-100mg of cholesterol is dissolved in
100ml of acetic acid.
5. Working standard Solution–4ml of stock solution is
dissolved in (or) diluted to 100ml with FeCl3-CH3COOH
solution. The concentration of standard is 0.04 mg/ml.

58. PROCEDURE :
STANDARDS :
1. Pipette ot 1-5 ml of standard solution in a series of testtubes.
2. The volume in each testtube is made upto 5ml with FeCl3-CH3COOH reagent.
3. 3ml of conc. H2SO4 is added to all the testtubes and mix well.
4. Standards are incubated for about 20-30 minutes at room temperature.
5. The intensity of standards is measured at 560 nm against blank.
BLANK :
5 ml of FeCl3-CH3COOH reagent, 3ml of H2SO4 are taken in a testtube, mixed
well and used as a blank.
TEST :
1. In the centrifuged tube 0.1ml of serum and 10ml of FeCl3-CH3COOH reagents
are taken, mixed well for 5 minutes and then centrifuged.
2. 5 ml of supernatant is collected and added with 3ml of H2SO4.
3. Test is incubated at room temperature to 20-30 Intensity is measured at 560nm
against blank.