CARBOHYDRATES

GLYCOGEN
• Principal storage form of carbohydrates in the mammalian body
• liver =6-8%
• muscles: -1%
• Major metabolic fuel for mammals and universal fuel for the
fetus
• Supply is necessary especially for the nervous system and
erythrocytes
• Good source of energy for sudden, strenuous activity
• A decrease in the blood glucose level below 70 mg/dL
(hypoglycemia) causes brain dysfunction which can lead to
coma and death
GLYCOGEN
Branched structure of glycogen
Muscle glycogen

 not generally available to other tissues because

muscles lack the enzyme glucose-6-phosphatase.
 provides a source of energy for muscle contraction
and is a readily available source of glucose for
glycolysis within the muscle itself.
 Glycogen is synthesized from glucose by the pathway
of glycogenesis, which occurs mainly in liver and
muscle.
 Glycogen synthase
 catalyzes formation of
glycosidic bonds between
C1 of the activated
glucose of UDPGlc and
C4 of a terminal glucose
residue at the non-
reducing end of glycogen,
liberating uridine
diphosphate (UDP)

Elongation of a glycogen chain by glycogen synthase.

 Glycogen synthase

 can only extend existing chain of glycogen.

 Glycogenin
 a.k.a. glycogen primer
 must be present to initiate glycogen biosynthesis
 37-kDa protein, which catalyses the addition of a glucose monomer to one of its own
tyrosine residues forming a bond between the C1 of glucose and the tyrosine hydroxyl
group.
 This reaction is due to glycogenin’s glucosyltransferase activity. Further glucose residues
can be added to the α1→4 position to make a short chain that is a substrate for glycogen
synthase.
 remains attached to the reducing end of the glycogen molecule.
 cannot make (α1→6) bonds found at the branch points of glycogen. Instead, these are
formed by a branching enzyme (amylo[1→4] →[1→6]-transglucosylase). When
the chain has been lengthened to at least 11 glucose residues, branching enzyme
transfers a part of the α1→4 chain (at least six glucose residues) to a neighboring
chain to form an α1→6 linkage, establishing a branch point. The branches grow by
further additions of α1→4-glucosyl units and further branching
 Glycogen synthase

 Glycogen is synthesized and degraded by different pathways.

The principal enzymes controlling glycogen metabolism
(glycogen phosphorylase and glycogen synthase) are regulated
by allosteric mechanisms and covalent modifications due to its
reversible phosphorylation and dephosphorylation in response
to hormones action (glucagon, insulin, epinephrine). Cyclic
adenosine-3’,5’- monophosphate (cAMP) integrates the
regulation of glycogenolysis and glycogenesis by promoting
the simultaneous activation of phosphorylase and inhibition of
glycogen synthase. Insulin acts reciprocally by inhibiting
glycogenolysis and stimulating glycogenesis.
Glycogen synthase
Isolation of liver glycogen
 Principle of the method :
 Glycosidic bonds in glycogen are resistant to hydrolytic
activity of OH- at elevated temperature. In contrast, peptide
bonds in proteins, ester bonds in lipids and phosphodiester
bonds in ribonucleic acids undergo hydrolysis at high
temperature and in alkaline pH (i.e. in KOH solution). Under
these conditions the glycogen solution, only slightly
contaminated with other polysaccharides, fragments of
denatured DNA and low molecular weight compounds, can
be obtained. Addition of ethanol results in glycogen
precipitation and allows to obtain relatively purified
glycogen.
 General properties of carbohydrates

Physical properties Relative Sweetness of sugars:

 Fructose ---------173.3%
carbohydrates Physical Solubility taste
appearance (water)  Invert sugar ----127-130%
 Sucrose ----------100%
Monosaccharid White crystalline soluble sweet
e  Glucose ----------74.3%
 Maltose ----------32.5%
Disaccharide White crystalline soluble sweet
 Lactose -----------16%
Starch Amorphous Slightly soluble tasteless
powder
cellulose fibrous Insoluble tasteless

CHEMICAL PROPERTIES:
THE ACTION OF NON OXIDIZING ACIDS
 With dilute acids, disaccharides and polysaccharides are hydrolyzed to monosaccharides.

In higher concentration (strong acids), such acids act on monosaccharides by

dehydrating (loss of water) them, forming furfural and furfural derivatives.
 GENERAL TESTS
FOR
CARBOHYDRATES
• Molisch test
• Anthrone’s test
• Iodine test
 Molisch test
 group test for all carbohydrates, whether free or in combined form.
 named after Hans Molisch, an Austrian botanist.

Principle:
The reaction is based on the fact that concentrated H2SO4
(dehydrating agent) catalyses the dehydration of sugars to form
furfural (from pentoses) or hydroxymethyl furfural (from
hexoses).These furfurals then condense with sulfonated alpha-
naphthol to give a purple or violet coloured ring at the junction.
Polysaccharides and glycoproteins also give a positive reaction. In
the event of the carbohydrate being a poly- or disaccharide, the acid
first hydrolyses it into component monosaccharides, which then get
dehydrated to form furfural or its derivatives.
PROCEDURE
1-2mL each of 1% carbohydrates sol’n

Add 2-3 gtt Molisch reagent

Mix
carbohydrates sol’n + Molisch reagent
Incline tube
Pour 1-2 mL conc. H2SO4

Formation of layers ( Violet ring at the junction)

ANTHRONE’S TEST

Principle:
Its principle is same as that for molisch’s test except
that the furfurals and hydroxy -methyl furfurals give
condensation products with anthrone that are bluish
green in colour.
PROCEDU
RE 1mL each of 1% starch sol’n

Add 2mL Anthrone reagent

starch sol’n + Anthrone reagent

heat
Note the change, cool

Bluish green color

Iodine test
 Principle:
The iodine test is used to test for the presence
ofstarch. Starch turns into an intense "blue-black"
colour upon addition of aqueous solutions of the
triiodide anion, due to the formation of an
intermolecular charge-transfer complex. In the
absence of starch, the brown colour of the aqueous
solution remains.
PROCEDURE
1mL each of 1% starch sol’n

Add few gtt of 0.01M Iodine sol’n

starch sol’n + iodine sol’n

heat
Note the change, cool

Bluish violet color

SPECIFIC TEST FOR
CARBOHYDRATES:
 Seliwanoff’s test  Barfoed’s test
 Bial’s Orcinol  Benedict’s test
 Mucic acid test  Tollen’s phloroglucinol test
 Osazone test  Tauber’s benzidine Test
 Iodine test
 Fehling’s test
 BASED ON THE FURFURAL FORMATION AND ITS
DERIVATIVES

SELIWANOFF’S TESTS
 for Keto Sugar

named after Theodor Seliwanoff

Principle:
This test is a timed colour reaction specific for ketohexoses. Thus it is used
to distinguish aldoses from ketoses. In the presence of HCl ketohexoses
undergo dehydration to yield 4-hydroxy methyl furfural more rapidly than
aldohexoses. Further these furfural derivatives condense with resorcinol to
form a red coloured complex.
PROCEDURE
1mL each of sugar sol’n

Add 5ml Seliwanoff’s reagent

sugar sol’n + Seliwanoff’s reagent

heat
Note the change for 15mins boiling

note color and time for each sol’n

 BASED ON THE FURFURAL FORMATION AND ITS
DERIVATIVES

BIAL’S ORCINOL
 Test for Pentoses

Principle:
This test is specific for pentoses and the compounds
containing pentoses and thus useful for the determination of
pentose sugars. Reaction is due to the formation of furfural in
the acid medium which condenses with orcinol in the presence
of ferric ions to give a blue green coloured complex.
PROCEDURE
o.5mL each of sugar sol’n

Add 4.5ml Orcinol reagent

sugar sol’n + Orcinol reagent

heat
Note the change for 15mins boiling

note color and time for each sol’n

BASED ON OXIDATION

MUCIC ACID TEST

Principle:
This test is highly specific for galactose which is either independently
present in solutions or obtained by the hydrolysis of lactose. Galactose is
converted to Saccharic acid on heating with HNO3 (a strong oxidizing agent).
Mucic acid (galactaric acid, a dicarboxylic acid) which is formed from galactose
due to the oxidation of both aldehyde & primary alcoholic group at C1&C6. It
is the only Saccharic acid which is insoluble in cold water and thus helps in the
identification of galactose.
PROCEDURE
50mg each of sugar sol’n

Add 1ml water+1 mL conc. HNO3

sugar sol’n + H2O+conc. HNO3

Heat (1hr)
Add 5mL H2O
Stand, cool slowly

No crystals crystals
Stand for next period Scratch crystals
of laboratory in test tube
crystals
Examine crystals under
microscope
BASED ON OXIDATION

OSAZONE TEST
 a.k.a PHENYLHYDRAZINE TEST OR KOWARSKY TEST

Principle:
An organic compound phenylhydrazine reacts with carbonyl carbon (or
more specifically C1 and C2) of sugar to form the compound called osazones
or phenylhydrazone. These osazone crystals have yellow colour
characteristics shapes and melting point, time of formation and solubility. All
reducing sugars form osazone with excess of phenylhydrazine
(C6H5NHNH2) when kept at boiling temperature. This property is attributed
to the presence of aldehyde or ketone group in their molecules.
PROCEDU
RE 5mL sugar sol’n

2ml Hot sol’n

Cover w/ cotton
Mix, heat (30mins)

Heated Sugar sol’n + hot sol’n

Cool at room temp
Examine crystals under microscope

Note time that crystals develop in each solution, draw

diff. Osazone
Under microscope =>
BASED ON OXIDATION

 Osazone test
 STAGES OF OSAZONE FORMATION:

A. The aldehyde group glucose interact with one molecule of phenylhydrazine

forming glucose phenylhydrazone

B. Phenylhydrazine, in excess reacts again with the hydrazone transforming the

alcohol group of the second carbon into a ketone

C. Finally, third molecule phenylhydrazine interacts with the newly formed ketone
group producing osazone.

 Similar stages occur in the case of fructose. The first molecule of

phenylhydrazine reacts with the carbon group. The second molecule oxidizes the
primary alcohol next to the carbonyl group and the third, combines with the
newly formed carbonyl group to form fructosazone.
 BASED ON OXIDATION

FEHLING’S TEST:
 A test to detect reducing and non-reducing sugar
Principle:
Sugar which contain a free aldehyde or ketone group
reduce alkaline solution of copper salts to cuprous oxide.
This test can be used to screen for glucose in urine, thus
detecting diabetes.
 BASED ON OXIDATION

FEHLING’S TEST:
Fehling’s Solution: Prepared by combining two separate solutions,
known as Fehling’s A and B. Fehling’s A which is deep blue. Fehling’s B
is a colorless solution. The copper (II) complex in Fehling’s solution is an
oxidizing agent and the reactive reagent in the test. The deep blue active
ingredient in Fehling’s solution in the bis(tartrate) complex of Cu2+. The
two solutions are not mixed until just before using, because the tartrate
itself would tend to reduce the copper in time. When a sugar solution
containing a free aldehyde or ketone group is boiled with Fehling’s
solution, the characteristic result in a brick-red precipitate of Cu2O.
Rochelle salt acts as chelating agent in this reaction:
 CuSO4 + 2KOH ------------ Cu(OH)2 + K2SO4
 2Cu(OH)2 + Reducing Sugar ------------ 2Cu2O + Aldonic acid
PROCEDU
RE 8 gtt of each sugar solution

Add 1 mL of mixed Fehling’s sol’n

sugar sol’n + Fehling’s sol’n

Boil (5mins)
Note the change

Brick red ppt

BASED ON OXIDATION

BARFOED’S TEST:
Principle:
This test is performed to distinguish between a
reducing mono- and disaccharide. Monosaccharides
are more reactive (easily oxidized) reducing agents
than disaccharides and thus react in about 1-2 min
while the reducing disaccharides take 7-12 min to get
hydrolysed in the acidic solution and then react. Thus,
the difference in reducing property can be detected.
PROCEDURE
10 gtt each of sugar sol’n + 3mL Barfoed’s reagent

Heat (15 mins)

cool
cool sugar sol’n+ Barfoed’s reagent mixture
Wait for 15 mins
Note the change after15mins

Brick-red ppt
BASED ON OXIDATION

BENEDICT’S TEST
Principle:
Benedict modified the Fehling’s solution to
produce an improved single reagent which quite
stable. Sodium Citrate functions as a chelating agent.
It is very sensitive and even small quantities of
reducing sugars(0.1%) yield enough precipitates.
PROCEDU
RE 2ml benedicts reagent + 5 gtt sugar sol’n
Heat (2 mins)

heated sugar sol’n , benedicts reagent mixture

cool
Note the change

Brick-red ppt
OTHER TESTS:
ACTION OF ACIDS
A. Tollen’s phloroglucinol test
o Test for galactose is also based on the formation
of similar intermediate furfurals which condenses
with phloroglucinol to form a red colored compound.
OTHER TESTS:
TEST FOR PENTOSES
A. Tauber’s benzidine Test
The production of violet color indicates the
presence of pentose.
OTHER SPECIFIC TEST FOR
CARBOHYDRATES
Iodine test
 for polysaccharides

 This test is performed to distinguish polysaccharides from mono- and

disaccharides.
Principle:
Iodine forms coloured adsorption complexes with different polysaccharides.
These complexes are formed due to the adsorption of iodine on the
polysaccharide chains. The intensity of the colour depends on the length of the
unbranched or linear chain available for the complex formation. Thus,
amylose, the unbranched helical component of starch gives a deep blue colour
and amylopectin, the branched component gives red colour because the chains
do not coil effectively. Glycogen, which is also highly branched, gives red
colour with iodine. This test is conducted in acidic or neutral solutions.
 General test for carbohydrates
Based on oxidation
Carbohydra Molish Anthrone’s Iodine test
Sugar sol’n Fehling’s Benedict’s Barfoed’s test Mucic acid te test test
test test test
Glycogen Violet ring Bluish green Yellow-
Glucose Brick red Brick Brick red ppt at the brown
ppt red ppt junction (maltose)
Fructose Brick red Brick Brick red ppt starch Violet ring Bluish Blue-violet
ppt red ppt at the green
junction
Galactose Brick red Brick Brick red ppt Needle-
ppt red ppt like
crystals
BASED ON THE FURFURAL
Maltose Brick red Brick Brick red ppt FORMATION AND ITS
ppt red ppt DERIVATIVES
Sucrose Blue Blue Blue
Sugar Seliwanoff’s Bial’s test
Lactose Brick red Brick Brick red ppt Needle- sol’n test
ppt red ppt like
crystals Glucose Yellow initially, Brown/
red w/ prolonged
heating for 15
dark red(-)
mins
Xylose Yellow initially, Blue
red w/ prolonged
BENEDICTS – is maintained in basic conditions heating for 15
green(+)
Barfoeds – is maintained in acidic condition mins
Fructose Cherry red or red Brown/
(immediately
dehydrated)
dark red(-)
Summary of theoretical result