What are Lipids ?

▪ Biological lipids are a chemically diverse group of compounds, and defining feature is they are
hydrophobic or amphiphilic (having both hydrophobic and hydrophilic parts).

▪ It is an Organic Compound that cannot form hydrogen bonds and thus insoluble in water and soluble
in non-polar solvents.

▪ Lipids include fats, oils, waxes, phospholipids, and sterols.

What are Fatty acids?

▪ Fatty Acids Are Hydrocarbon Derivatives

▪ Fatty acids are carboxylic acids with hydrocarbon

chains ranging from 4 to 36 carbons long (C4
to C36).

▪ In some fatty acids, this chain is unbranched

or branched.

▪ Fully saturated (contains no double bonds);

▪ OR contains one or more double bonds.

▪ Fatty acids are also called as FATS.
▪ Mostly even C containing.
▪ Essential (L, LN) or
▪ Non-essential F.A.
 STRUCTURES

▪ The most significant property of lipids

are their hydrophobic properties, which
are mostly because of Fatty acids.
Nature of lipid molecules:

Length of Fatty acid chain Saturated/ Unsaturated Fatty acid

Shorter Longer Saturated Unsaturated Fatty acid

chain length chain length

More compact structure

Less interaction More interaction Less compact structure
More interaction

Less interaction
Less fluidity (rigid structure)
More Fluidity Less Fluidity

More fluidity (fluid structure)

 Movement of lipid and Membrane Fluidity
➢Interaction between lipid in single lipid layer- Vanderwall’s force
of interaction
➢Interaction between lipid in lipid bilayer- Hydrophobic interaction

Movement of lipid Molecules-

Rotational
Lateral movement
Flip- flop movement
1. Rotational movement
• spin along axis
• less frequent
• passive
• no linkage with fluidity
2. Lateral movement
• movement of lipid within lipid layer
• more frequent
• passive
• ATP independent
• linkage with fluidity (inversely prop)
 NOMENCLATURE OF THE FATTY ACIDS

▪ A simplified nomenclature for unbranched fatty acids specifies the chain

length and number of double bonds, separated by a colon.

▪ For example, the 16-carbon saturated palmitic acid is abbreviated 16:0, and
the 18-carbon oleic acid, with one double bond, is 18:1.
 TWO CONVENTIONS FOR NUMBERING FATTY ACIDS

(a) Standard nomenclature assigns the number 1 to the carboxyl carbon (C-1),
and α to the carbon next to it.

zigzag line segment represents a single bond between adjacent carbons.

The Double bond(s) is indicated by ∆ followed by a superscript number

indicating the lower-numbered carbon in the double bond.
A PUFA, 20-carbon fatty acid with one double bond between C-9 and C-10 (C-1 being
the carboxyl carbon) and another between C-12 and C-13 is designated 20:2 (∆ 9,12).

In most monounsaturated fatty acids the double bond is between C-9 and C-10 (∆9),
and the other double bonds of polyunsaturated fatty acids are generally ∆12 and ∆15.
(b) For polyunsaturated fatty acids (PUFAs), an alternative convention
numbers the carbons in the opposite direction, assigning the number 1 to the
methyl carbon at the other end of the chain; this carbon is also designated “ꙍ "
(omega; the last letter in the Greek alphabet).

▪ The positions of the double bonds are indicated relative to the ꙍ carbon.
 Polyunsaturated fatty acids (PUFAs)

Omega-3 (ω-3) and Omega-6 (ω-6) fatty acids are both types of polyunsaturated
fatty acids (PUFAs), meaning they contain multiple double bonds in their carbon
chains.

What distinguishes them from each other is the position of the first double bond
relative to the methyl (omega) end of the molecule.

Omega-3 Fatty Acids:

• The first double bond is located at the third carbon from the methyl end of the fatty
acid chain.

• Examples of omega-3 fatty acids:Alpha-linolenic acid (ALA), Eicosapentaenoic

acid (EPA), Docosahexaenoic acid (DHA).

Omega-6 Fatty Acids:

• The first double bond is located at the sixth carbon from the methyl end of the fatty
acid chain.

• Examples of omega-6 fatty acids: Linoleic acid (LA), Arachidonic acid (AA).
▪ In nearly all naturally occurring unsaturated fatty acids, the double bonds are in the cis
configuration.

In cis fatty acids, the hydrogen atoms attached

to the carbon atoms are on the same side of the
carbon chain.

▪ This configuration causes a bending or

kink in the carbon chain.

▪ Prevents the fatty acids from packing

tightly together, leading to liquid form at
room temperature.

▪ all naturally occurring unsaturated fatty

acids, the double bonds are in the cis
configuration.

▪ Olive oil
Trans Fatty Acids:

• In trans fatty acids, the hydrogen atoms

attached to opposite sides of the carbon chain.

• This configuration results in a straighter

carbon chain.

• trans fats behave more like saturated fats.

• The straight shape allows trans fats to pack

tightly together, which often results in a solid
form at room temperature

• This is why trans fats are commonly found in

processed foods.
▪ Trans fatty acids in the diet are
• Examples: Trans fats are often artificially an important risk factor for
created during the process of hydrogenation, coronary heart disease.
used to solidify liquid vegetable oils.
 FATTY ACID COMPOSITION OF
THREE FOOD FATS

o Olive oil, butter, and red meat fat consist of

mixtures of triacylglycerols, differing in their
fatty acid composition.

o The melting points of these fats, their

physical state at RT are a direct function of
their fatty acid composition.

o Olive oil has a high proportion of long-chain

(C16 and C18) unsaturated fatty acids,
which accounts for its liquid state at 25ᵒC.

o Butter: The higher proportion of long-chain

(C16 and C18) saturated fatty acids in butter
increases its melting point, so butter is a
soft solid at RT.

o Red meat fat, with an even higher

proportion of long-chain saturated fatty
acids, is a hard solid.
o The melting points of these FATTY ACID COMPOSITION OF
fats, their physical state at THREE FOOD FATS
RT are a direct function of
their fatty acid composition.
 Biological Functions of Lipids

a. Storage of energy – reduced compounds: lots of available energy – hydrophobic nature:

good packing

b. Insulation from environment – low thermal conductivity – high heat capacity (can
“absorb” heat) – mechanical protection (can absorb shocks)

c. Water repellant – hydrophobic nature: keeps surface of the organism dry

d. Prevents excessive wetting (birds)

e. Prevents loss of water via evaporation

f. Acts as structural components of cell membranes

g. Eicosanoids: These lipid mediators include prostaglandins, leukotrienes,

and thromboxanes, which play roles in inflammation, blood clotting, and immune
responses.

h. Acts as precursor for synthesis of pigments…….

Fats are solid, while oils are liquid. •Fats: There are two main types of fats:
Source: Fats are mainly animal-derived, while • Saturated fats: These are solid at
oils are mainly plant-derived. room temperature. Examples include
Composition: Fats are composed of saturated butter, beef fat, and cream.
fats, while oils contain unsaturated fats. • Trans fats: These are also solid fats
Health impact: Unsaturated fatty acids in oils and are found in cookies, chips, and
do not raise cholesterol levels and can help processed foods. It’s best to
lower LDL (bad) cholesterol levels. consume fewer foods containing
trans fats due to their impact on
cholesterol levels.
Caloric Content: Both fats and
oils contain 9 calories per gram.
•Oils: Oils are liquid at room temperature and fall into
two categories:
• Monounsaturated fats: Found in nuts, vegetable
oils, and avocados. Consuming foods rich in
monounsaturated fats helps control cholesterol
levels.
• Polyunsaturated fats: Found in oils like
sunflower, corn, and soybean. Seafood is a major
source of these fats. Replacing saturated fats
with polyunsaturated fats may help lower LDL
cholesterol
 1. STORAGE LIPIDS

▪ Fats and Oils are universally used as stored form of energy. These are derivatives of Fatty
acids

▪ Cellular oxidation of Fatty acids is a highly exergonic process. P.A=106

Triacylglycerols or Triglycerides: Neutral lipids.

✓ The simplest lipids constructed from fatty acids are the

triacylglycerols. Exists in two forms- fats, or oil.

✓ Triacylglycerols are composed of three fatty acids each in

ester linkage with a single glycerol.

✓ Triacylglycerols Provide Stored Energy and Insulation.

 TRIACYLGLYCEROLS

▪ Are Fatty Acid Esters of Glycerol

▪ Simplest lipids.

▪ Also referred to as triglycerides, fats, or neutral fats.

▪ Composed of three fatty acids each in ester linkage with a

single glycerol.

▪ Triacylglycerols are primarily storage fats;

▪ In vertebrates, adipocytes, or fat cells, stores triacylglycerols

as fat droplets.

▪ In the seeds of many types of plants, stored as oils providing

energy and biosynthetic precursors during seed germination.

▪ Adipocytes and germinating seeds contain lipases, enzymes

that catalyze hydrolysis of stored triacylglycerols.
Cotyledon cell from a seed of Arabidopsis
 Two significant advantages to using triacylglycerols as stored fuels:

(1) the carbon atoms of fatty acids are more reduced than those of sugars, and oxidation of triacylglycerols yields more than twice
as much energy.

(2) Triacylglycerols are hydrophobic and therefore unhydrated, the organism that carries fat as fuel does not have to carry the extra
weight of water of hydration.
 WAXES

▪ Serve as Energy Stores and Water Repellents.

▪ Biological waxes are esters of long-chain (C14 to C36) saturated and

unsaturated fatty acids with long-chain alcohols.

▪ Insoluble and have high melting points.

Variety of functions:

✓ Storage of metabolic fuel in plankton

✓ Protection and pliability for hair and skin in vertebrates

✓ Waterproofing of feathers in birds

✓ Protection from evaporation in tropical plants and

✓ Used by people in lotions, ointments, and polishes

Wax: the material of the honeycomb.

Beeswax,is a mixture of a large number of lipids, including esters of triacontanol,

and a long-chain palmitic acid.
 2. STRUCTURAL LIPIDS IN MEMBRANES

▪ The central architectural feature of biological membranes is a double layer of lipids,

which acts as a barrier to the passage of polar molecules and ions.

▪ Present in bilayer form.

▪ Amphipathic (hydrophilic and hydrophobic).

Some common types of storage and membrane lipids.

 Glycerophospholipids Are Derivatives of Phosphatidic Acid

Glycerophospholipids, also called phosphoglycerides, are membrane lipids in which two

fatty acids are attached in ester linkage to the first and second carbons of glycerol.
 • Phospholipids or Glycerophospholipids, also called phosphoglycerides.
Lipids containing, in addition to fatty acids and an alcohol, a phosphoric acid
residue.

• Glycolipids (glycosphingolipids): Lipids containing a fatty acid, sphingosine,

and carbohydrate.
 Some Glycerophospholipids Have Ether-Linked Fatty Acids

Some animal tissues and some unicellular organisms are rich in ether lipids, in which one of
the two acyl chains is attached to glycerol in ether, rather than ester, linkage.
 Archaea contain unique membrane lipids

▪ Most of the archaea live in ecological extreme conditions (e.g. high temperatures, low pH,
high ionic strength).

▪ The have membrane lipids containing long-chain branched hydrocarbons linked at each
end to glycerol through ether bonds.

▪ Ether bonds are more stable to hydrolysis at low pH and high temperatures.

▪ These archaea lipids are twice the length of phospholipids and sphingolipids, and can
span the full width of the surface membrane.
 Sphingolipids Are Derivatives of Sphingosine

▪ Sphingolipids, the fourth large class of membrane lipids, also have a polar
head group and two nonpolar tails.

▪ The backbone of sphingolipids is NOT glycerol. The backbone of

sphingolipids is a long-chain amino alcohol, sphingosine.

▪ A fatty acid is joined to sphingosine via an amide linkage rather than an ester
linkage as usually seen in lipids.

▪ A polar head group is connected to sphingosine by a glycosidic or

phosphodiester linkage.

▪ The sugar-containing glycosphingolipids are found largely in the outer face of

plasma membranes.
 Three subclasses of sphingolipids

(1) Sphingomyelins contain phosphocholine or phosphoethanolamine as their

polar head group and are therefore classified along with glycerophospholipids
as phospholipids. Sphingomyelin is abundant in myelin sheath that surrounds
some nerve cells in animals

(2) Glycosphingolipids which occur largely in the outer face of plasma

membranes, have head groups with one or more sugars connected directly to
the ─OH at C-1 of the ceramide moiety.

(3) Gangliosides the most complex sphingolipids, have oligosaccharides as

their polar head groups and one or more residues of N-acetylneuraminic acid
(Neu5Ac), a sialic acid, at the termini.
GLYCOSPHINGOLIPIDS AS DETERMINANTS
OF BLOOD GROUPS

▪ The human blood groups (O, A, B) are determined

in part by the oligosaccharide head groups of
these glycosphingolipids.

▪ The same three oligosaccharides are also found

attached to certain blood proteins of individuals of
blood types O, A, and B, respectively.
 Phospholipids and Sphingolipids Are
Degraded in Lysosomes

▪ Most cells continually degrade and

replace their membrane lipids.

▪ For each hydrolysable bond in a

glycerophospholipid, there is a
specific hydrolytic enzyme in the
lysosome.
 3. STEROLS

• Consisting of four fused rings, three with six carbons and one with five.

• Similar sterols are found in other eukaryotes: stigmasterol in plants and

ergosterol in fungi.
Bile acids are polar derivatives of cholesterol that act as detergents in the intestine,
emulsifying dietary fats to make them more readily accessible to digestive lipases.
 LIPIDS AS SIGNALS, COFACTORS, AND PIGMENTS

▪ Some types of lipids, play critical roles as cofactors or signals.

▪ Phosphatidylinositol 3,4,5-trisphosphate (IP3) acts as intracellular

messengers involved in biological signaling.

▪ Prostaglandins, thromboxane’s and leukotrienes, derived from

arachidonate, are extremely potent hormones.

▪ Steroid hormones, such as the sex hormones, are derived from sterols. They
serve as powerful biological signals, altering gene expression in target cells.
vitamins d, a, e, and k are fat-soluble compounds made up of isoprene units.

all play essential roles in the metabolism or physiology of animals.

vitamin d is precursor to a hormone that regulates calcium metabolism.

vitamin a furnishes the visual pigment of the vertebrate eye and is a regulator of
gene expression during epithelial cell growth.

vitamin e functions in the protection of membrane lipids from oxidative damage,

and vitamin k is essential in the blood-clotting process.

ubiquinone's and plastoquinone's, also isoprenoid derivatives, are electron carriers

in mitochondria and chloroplasts, respectively.

lipidic conjugated dienes serve as pigments in flowers and fruits and give bird
feathers their striking colors.

polyketides are natural products widely used in medicine.

 Common procedures in the extraction, separation, and
identification of cellular lipids

Lipid Extraction Requires Organic Solvents

Neutral lipids (triacylglycerols, waxes, pigments, and so

forth) are readily extracted from tissues with ethyl ether,
chloroform, or benzene, solvents that do not permit lipid
clustering driven by hydrophobic interactions.

(a) Tissue is homogenized in a chloroform/

methanol/water mixture, centrifugation yields two
phases.

(b) Major classes of extracted lipids in the chloroform

phase may first be separated by TLC.

(c) Mass spectrometry is done to determine the total

composition of all the lipids: the lipidome.
 QUALITATIVE ANALYSIS OF FATS

1) SOLUBILITY TEST

Solubility test is the preliminary test which detects the presence of all lipids. This test
detects the solubility of lipid in various solvents to check whether it is miscible or
immiscible in polar or non-polar solvents.

Principle: Solubility test is based on the property of lipid to dissolve in different solvents.
Lipids are readily miscible in non-polar solvents like chloroform, partially soluble in a polar
solvent like ethanol and immiscible in a polar solvent like water.

Method:

i. Take the sample of lipid in three different test tubes by labelling it as A, B and C.
ii. Then add different solvents like water, ethanol and chloroform in three different test
tubes.
iii. Shake the tubes and allow it to stand for 1 minute.
2) TRANSLUCENT SPOT TEST:

A translucent spot test is also a preliminary test for the lipids which can be detected by the
appearance of a translucent and greasy spot.

Principle: The lipid will not wet the filter paper, unlike water. The lipid will form a greasy
spot as they are having a greasy texture that will penetrate into the filter paper. In contrast to
lipid, the spot of water will disappear from the paper whereas the spot of lipid appears as the
“Translucent spot”.

Method:
• Take a filter paper.
• Add one drop of water on one end and a drop
• of oil or lipid on the other end.
 3. SAPONIFICATION TEST: It is based on the
saponification reaction, in which the triglycerides of
lipid react with an alkali NaOH to produce salt of fatty
acids/soap and glycerol in the presence of ethanol.

✓ This reaction is also known as alkaline hydrolysis

of esters.

4. DICHROMATE TEST:

✓ Dichromate test is also used to detect the presence of

glycerol. It is based on the principle of an oxidation
reaction.

✓ In this, glycerol and dichromate ions react to give a

brown colour to the solution.

✓ Then, the chromic ions oxidize the glycerol and reduce

into chromous ions by giving a blue colour to the
solution in the presence of nitric acid.
 5. ACROLEIN TEST

Principle: When a glycerol derived fat is heated strongly in the presence of a dehydrating
agent such as potassium bisulfate (KHSO4). The glycerol portion of the molecule is
dehydrated to form the unsaturated aldehyde, ACROLEIN (CH2=CH–CHO), which has
the odour peculiar to burnt cooking grease.

Note: Take pure glycerol as positive control and oil sample in a dry test tube; add to it a
few crystals of potassium hydrogen sulphate. Warm gently to mix and then heat strongly. A
very pungent odour of acrolein is produced. Acrolein is formed due to removal of water
from glycerol by potassium hydrogen sulphate.
 6. SUDAN III TEST

▪ Sudan III is a red fat-soluble dye that is used in the identification of the
presence of lipids, triglycerides and lipoproteins which relies on hydrophobic
interactions between Sudan III dye and lipids.

▪ Sudan III dissolved in ethanol is allowed to interact with the lipids bound to a
filter, then when the filter is washed with water the water will not permit Sudan
III bound to the lipids to escape. Consequently, spots containing lipids will
appear orange against a pink background.
 7. CHOLESTEROL

Salkowski test: Is used to detect Cholesterol in solution. Test is named after German
Biochemist Ernst Leopold Salkowski.

Principle: Sterols are alcohols, the Salkowski test involves treating sterols like cholesterol
with a strong acid (are hygroscopic) which leads to the protonation of the hydroxyl groups
of the alcohol that leads to a DEHYDRATION REACTION. H2SO4 removes two molecules
of water from two molecules of cholesterol.

i. CONDENSATION of two sterols: Two sterols bind together at position 3, forming bi-
cholestadien (double cholestene with two double bonds) in a case of cholesterol.

ii. Simultaneously H2SO4 SULPHONATES bi-cholestadiene giving red color.

Method:

1. Add 2 ml of the provided chloroform to cholesterol.

2. Add an equal volume of concentrated sulfuric acid

(H2SO4).

3. Observe the color in varying concentration of

Cholesterol.

A positive test is indicated by: A yellow to brick-red

color is formed indicating the presence of cholesterol.
 Iodine test

Principle: This test is used to test the degree of unsaturation of fatty acids.

▪ Fatty acids in animal fats are usually saturated, whereas those in vegetable oils
are generally unsaturated.

▪ Halogens like iodine or bromine when added to unsaturated fatty acid the double
bond will be saturated and decolorize the iodine or bromine, the decolorization
indicates the presence of unsaturated fatty acids.

▪ Iodine test is used for distinguish between saturated and unsaturated fatty
acids as well as between oils and fats.
Lipid Extraction, Separation and Analysis:
The principle physiochemical characteristics of lipids used to distinguish
them from the other components in foods are:

✓ Their solubility in organic solvents

✓ Physical characteristics(e.g; relatively low density) and

✓ Spectroscopic properties.

The Analytical techniques based on these principles can be categorized into

three different types:

1. Solvent Extraction Method

2. Non-solvent Extraction Method

3. Bligh & Dyer

4. Folsc’h Method

5. Instrumental Methods
 LIPID EXTRACTION METHODS:

➢ Lipids are soluble in organic solvents but insoluble in water, hence

their extraction requires:

1. SOLVENT EXTRACTION METHOD:

➢ It is the most commonly used method for extraction of lipids from

both biological samples or food samples.

➢ The weight of the lipid is measured by: Weighing the extracted lipid
sample after evaporating the organic solvent.
 STEPS IN SOLVENT EXTRACTION METHOD:

1. Sample Preparation.
2. Solvent Extraction Methods.

1. SAMPLE PREPARATION: The preparation of a sample for solvent

extraction involves number of steps:

i) Drying sample: It is often necessary to dry samples prior to solvent

extraction, because many organic solvents cannot easily penetrate into foods
containing water, and therefore extraction would be inefficient.

ii) Particle size reduction: Is done to increase adsorption of solvent by

lipid. Grinding is done to reduce particle size.

iii) Acid hydrolysis: Some foods contains lipids that are complexed with
protein or polysaccharides, so sample is digested by heating it for 1hr in the
presence of Hcl.

iv) Solvent selection: Choose the best solvent for the extraction.
Ideal solvent for lipid extraction must have:

✓ High solvent power for lipids

✓ Low solvent power for non-lipids

✓ Evaporate easily

✓ Non flammable

✓ Non-toxic

✓ Cheap
Solvent Selection:

✓ Polar lipids (such as glycolipids or phospholipids) are more

soluble in polar solvents (such as alcohols), than in non-polar
solvents (such as hexane).

✓ On the other hand, non-polar lipids (such as triacylglycerols) are

more soluble in non-polar solvents than in polar ones.

✓ The fact that different lipids have different polarities means that it
is impossible to select a single organic solvent to extract them all.
So more than one solvent is required for lipid extraction.

✓ Ethyl ether and petroleum ether are the most commonly used
solvents, but pentane and hexane are also used for some foods.
Ethyl ether is used but is: Ethyl ether is an excellent solvent
for lipids:
✓ Very flammable
✓ More selective for more
✓ Explosion hazard hydrophobic lipids

✓ Forms peroxides ✓ Cheaper

2. NON-SOLVENT LIQUID EXTRACTION METHODS:

➢ A number of lipid extraction methods do not rely on

organic solvents, but use other chemicals to separate
the lipids from the rest of the food.

➢ These are the non-solvent liquid extraction methods

for determining the lipid content of milk and some
other dairy products:

i) The Babcock Method.

ii) The Gerber Method and

iii) Detergent method.

i) Babcock Method: ii) Gerber Method:
➢ A specified amount of milk is ➢ This method is similar to the
accurately pipetted into a Babcock method except that:
specially designed flask (the
Babcock bottle). ✓ A mixture of sulfuric acid
and isoamyl alcohol, and
Principle: Milk + Sulphuric
acid. ✓ A slightly different shaped
bottle, are used.
Sulfuric acid causes digestion of
protein and breaks down the fat
globule membrane that
surrounds the droplets, thereby
releasing the fat.
iii) 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 detergents will displace the fat

globule membrane which surrounds
the emulsion droplets in milk and
causes them to 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.
 Total Lipid Extraction Methods:
➢ The two most conventional methods of lipid extraction are, namely, Folch
method and Bligh and Dyer method.

➢ In both the methods, solvents such as chloroform and methanol are used
in the ratio 2:1 and 1:2 v/v, respectively.

1. Folch Method:

Principle: Lipids are extracted by chloroform–methanol mixture (2:1 by

volume). Extract is washed from water soluble impurities, dried and then
obtained precipitate is weighed.

➢ Briefly, the homogenized cells were equilibrated with one-fourth volume of

saline solution and mixed well.

➢ The resulting mixture was allowed to separate into two layers and lipids
settle in the upper phase.
 Bligh and Dyer Method:

➢ Lipid extraction and partitioning are performed simultaneously in

the Bligh and Dyer (1959) method.

➢ The Bligh and Dyer method is one of the widely practiced methods for
lipid extraction. It is very similar to the Folch method, but mainly
differs in solvent/solvent and solvent/tissue ratios.

➢ This procedure is performed by extracting lipids from homogenized

cell suspension using 1:2 (v/v) chloroform/methanol.

➢ The lipids from the chloroform phase are then extracted and
processed by various procedures.

➢ The above gravimetric method is still widely used for the estimation of
lipids by algal technologists.
CALCULATIONS:

Use the absorbance readings of the STANDARD and UNKNOWN(S)

to calculate total lipid values as follows:

A = Absorbance

Total Lipid in UNKNOWN (mg/dl) = A (UNKNOWN) x Conc. of STD

A (STANDARD) (mg/dl)

Example: A (patient) = 0.45

A (standard) = 0.50
Concentration of standard = 600 mg /dl

0.45 x 600 mg/dl = 540 mg/dl 0.5