[Carbohydrate] Biochemistry

Carbohydrates
1. Introduction:
Carbohydrates are the compounds which contain carbon, hydrogen & oxygen. They are
commonly known as saccharides or sugars. Carbohydrates are widely distributed in nature in
plants and animals. The most common carbohydrate found in plants is Starch. The carbohydrate
found in animal is Glycogen. It is a storage form of carbohydrates found in liver and muscles.
Chemical composition: carbohydrate have an empirical formula Cn(H2O)n.
Definition:
Chemically carbohydrates may be defined as polyhydroxy aldehydes or ketones and their
substance which yield on these substances on hydrolysis.
Role of Carbohydrates:
 Carbohydrates are the chief source of energy.
 They form the constituent of various biological compounds like as lipids and conjugated
proteins.
 Some carbohydrates derivative is used as drug like a cardiac glycosides and antibiotics.
 Lactose is principal sugar in milk.

2. Classification of carbohydrates:
Carbohydrates are classified into following groups:
I. Monosaccharides.
II. Disaccharides.
III. Oligosaccharides.
IV. Polysaccharides.

I. Monosaccharides:
They contain only one molecule of sugar. They are the simplest carbohydrates so called
simple sugars. They have the general formula (CH 2O)n. e.g.:- Glucose and Fructose.
They are further classified as follow:

Trioses- containing 3 carbon atoms.

Tetrose- containing 4 carbon atoms.
Pentoses- containing 5 carbon atoms.
Hexoses- containing 6 carbon atoms.

II. Disaccharides:
Disaccharides yield two molecules of Monosaccharides on hydrolysis. They have the
general formula Cn(H2O)n-1.
Example: Sucrose, Maltose and Lactose.

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III. Oligosaccharides:
They produce two to ten molecules of Monosaccharides on hydrolysis. The general
formula of Tetrasaccharides Cn(H2O)n-2. e.g.:- Raffinose.
The general formula of Pentasaccharides Cn(H2O)n-3. e.g.:- Stachyose.

IV. Polysaccharides:
They produce more than ten molecules of Monosaccharides on hydrolysis. They have
general formula C6H10O5. e.g.:- Starch, glycogen, dextran and cellulose.
Polysaccharides are further classified as follow:

I. Homopolysaccahride: If they yield only one type of monosaccharide unit on hydrolysis.

Example: Starch, Glycogen and Cellulose etc.

II. Heteropolysccharide: If they yield more than one type of monosaccharide unit on hydrolysis.
Example: Heparin and Hyaluronic acid.

3. Identification test for Carbohydrates:

I. Molish’s Test: mix 2 ml of carbohydrate sample with 5 drops of Molish’s reagent in a

test tube. Add 2 ml of concentrated Sulphuric acid so as to form a bottom layer.
Observation: violet/purple ring at the function of two liquids.
Inference: carbohydrate present.

II. Solubility Test: compound + water

Observation: soluble/insoluble
Inference: Mono and Disaccharides present (soluble).
Polysaccharides present (insoluble).

III. Fehling’s Test: 2 ml of Fehling’s solution A + 2 ml of Fehling’s solution B + 2 ml of

sugar solution.
Observation: yellow or brick red colored ppt is observed.
Inference: reducing sugar is present.

IV. Benedict’s Test: Take 5 ml of Benedict’s test solution; add 8 drops of sugar solution.
Boil over a flame for 2 minutes or place in boiling water bath for 3 minutes. Allow to
cool.
Observation: green yellow, orange or brick red ppt is observed.
Inference: reducing sugar is present.

V. Tommer’s Test: To 2 ml of tommer’s reagent add 3 ml of sugar solution boil for 2

minutes and then cool.
Observation: yellow or red ppt is observed.
Inference: reducing sugar present.

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VI. Barfoed’s Test: 2 ml of Barfoed’s test reagent add 2 ml of sugar solution and keep in
boiling water bath for 2 minutes and cool it.
Observation: Brick red ppt is observed at the bottom of test tube.
Inference: Reducing sugar present.

VII. Seliwanoff’s Test: To 3 ml of Seliwanoff’s reagent add 1 ml of sugar solution and heat
the mixture to boil for 2 minutes and the cool it.
Observation: red Colour or red ppt is observed at the bottom of test tube.
Inference: Ketoses like Fructose, sucrose are present.

VIII. Rapid furfural Test: to 2 ml of sugar solution add 1 ml of α-napthol solution (1% in
alcohol) and 5 ml concentrated Hcl and boil it.
Observation: Deep purple colour is observed.
Inference: Ketoses like Fructose, Sucrose are present.

IX. Osazone Test: set up a boiling water bath, take test tube, and add 1 gm of phenyl
hydrazine hydrochloride. 2 ml of acetate buffer pH 5.0 add 5 ml of water mix well and
warm gently. Filter it. To the filtrate add 5 ml sugar solution and keep in boiling water
bath and cool. Mount the crystals under microscope and examine under microscope.

Observation: Greenish yellow needle shaped crystals.

Badminton ball, powder puff shaped crystals
Sunflower or its petal shaped crystals.

Inference: Glucosazone i.e., Glucose present.

Lactosazone i.e., Lactose present.
Maltosazone i.e., Maltose present.

X. Iodine test:
a) To about 2 ml suspension or solution of polysaccharides add 1-2 drops of N/50 iodine
solution.
b) Warm above solution
c) Cool above solution
d) To about 2 ml suspension of starch adds 1 ml of 5% sodium hydroxide & 2 drops of
N/50 iodine.

Observation:
a) Blue violet colour develops. Brown wine colour develops
b) Blue colour disappear
c) Blue colour reappear
d) No Blue colour
Inference:
a) Starch is present, glycogen is present
b) Starch is present.
c) Starch is present
d) Starch is present

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3.1.Common test for glucose:

a) Osazone test
Observation: greenish yellow needle shaped crystal.
Inference: Glucose confirmed

b) To 2 ml of test solution add 5% NaOH.

Observation: brown resinous precipitate is observed.
Inference: Glucose confirmed

c) Take 3 ml water to it add drop of methylene blue solution and 1 ml of 5% sodium hydroxide and
2 ml of sugar solution and boil it.
Observation: solution is decolorized.
Inference: Glucose confirmed

d) To 3 ml of sugar solution add 1 ml of picric acid solution and 1 ml of 5 % NaOH and supply
heat.
Observation: Red color develops.
Inference: Glucose confirmed

3.2.Common test for Lactose:

1. Mucic acid Test:

To 1ml sugar solution adds 1ml conc. HNO3 and boils it. Cool the boiled solution.

Observation: Broken glass like crystals obtained which can be identified under microscope.
Inference: lactose confirmed.

2. Silver Mirror Test:

1 ml sugar solution add 1ml Ammoniacal silver nitrate solution add excess NaOH warm it.

Observation: silver mirror is observed.

Inference: lactose is confirmed.

3.3.Common test for Sucrose:

a) Seliwanoff’s test
b) Rapid furfural test
c) Inversion test: to 25 ml of test solution add 5 ml of concentrated Hcl boil for 3 minutes
and cool under tape water. Neutralize with sodium hydroxide solution. Now perform
following test for this solution:

I. Fehling’s test.
II. Tommer’s test.
III. Benedict’s test.
IV. Barfoed’s test
Observation: Red precipitate is observed.
Red precipitate is observed.

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Red/Yellow/ Green precipitate is observed.

Inference: Sucrose confirmed.

Sucrose confirmed
Sucrose confirmed

3.4.Common test for Fructose:

Fougler’s test:
To 3 ml Fougler’s reagent add 0.5 ml of sugar solution. Boil for one minute.

Observation: Blue colour develops.

Inference: Fructose confirmed.
4. Chemical Properties of Carbohydartes:

I. Tautomerization (Enediol formation):

In mild alkaline solution, carbohydrates containing free aldehyde and keto group will
tautomerise and produce enediols, in which two hydroxyl groups are attached to the double-
bonded carbon in mild alkaline conditions; glucose is converted into fructose and mannose.
O OH
H-C-OH

H OH C-OH O
HO H HO H
HO-C-H
H OH H OH
H OH H-C-OH
H OH
H-C-OH
OH OH
CH2OH
D-Glucose 1-2 Enediol D-Fructose
II. Benedict’s Reaction
It contains sodium carbonate, copper sulphate and sodium citrate. In alkaline medium,
sugars produce enediols which reduce cupric ions and respectively the sugar is oxidized. Sugar
with free aldehyde or keto group reduce Benedict’s reagent and termed as reducing sugars.

H O -
O O
2-
C + Cu (Citrate)2 C + Cu2O (s)

R R
An Aldose Benedicts's Reagent Carboxylate anion Brick red colour
Blue solution

III. Osazone formation:

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All reducing sugars form osazone with excess of phenylhydrazine when keep at boiling
temperature. Osazones are insoluble. Each sugar has characteristic crystal form of osazone

CHO CH=NNHC6H5 CH=NNHC6H5

H-C-OH C6H5NHNH2 H-C-OH H-C=NNHC6H5

2C6H5NHNH2
HCL
(CHOH)4 (CHOH)4 (CHOH)4

CH2OH CH2OH CH2OH

D-Glucose D-Glucose Phenyl hydrazine D-Glucosazone

IV. Oxidation of Sugars

In mild oxidation conditions the aldehyde group is oxidized and converted to carboxyl
group which produce aldonic acid. Ex: Glucose produce Gluconic acid. While aldehyde group is
secured and the molecule is oxidized the last carbon becomes COOH group and produce uronic
acid. Ex: Glucose to Glucuronic acid.
COOH COOH

H-C-OH H-C-OH

(CHOH)4 (CHOH)4

CH2OH COOH

D-Gluconic acid D-Glucoronic acid

In strong oxidation conditions the first and last carbon atoms are concurrently oxidized to
produce dicarboxyllic acids which are known as saccharic acids. Ex: Glucose converts to
Glucosaccharic acid.

V. Furfural Derivatives
When monosaccharides treated with concentrated sulphuric acid they undergo
dehydration by removal of three molecules of water. Then hexoses produce hydroxymethyl
furfural and pentoses produce furfural. These furfural derivatives may condense with phenolic
compounds to produce coloured products. This is the base for Molisch test, a general test for
carbohydrates.

VI. Reduction to produce Alcohols

When monosaccharide treated with reducing agents such as sodium amalgam, hydrogen
may reduce sugars. Aldose produces equivalent alcohols. But ketose produces two alcohols,

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because of presence of a new asymmetric carbon atom. Ex: Glucose produce sorbitol and
fructose produce sorbitol and mannitol.
CH2OH

H-C-OH

(CHOH)4

CH2OH
Glucitol or Sorbitol
(a sugar alcohal)

5. Disease Related to Carbohydrates Metabolism:

Disease related to carbohydrate metabolism is as follow:
I. Diabetes mellitus (hyperglycemia)
II. Hypoglycemia
III. Glycosuria
I. Diabetes mellitus (hyperglycemia:

Diabetes Mellitus is a disorder caused due to deficiency of insulin, which shows

clinically as a high blood glucose. It has been classified as per old classification system that is
based on a patients’ dependence on insulin was confusing; as per old system patients were either
classified as either Insulin Dependent Diabetes Mellitus (IDDM) or Non-Insulin Dependent
Diabetes Mellitus (NIDDM).
In 1998, a new classification system based on the etiological factors was proposed by the
WHO and listed below: this has now become the accepted system for classifying diabetes
mellitus.

Type 1 diabetes:
Type 1 diabetes is immune mediated and idiopathic forms of β-cell dysfunction, which
lead to insulin deficiency. This is an autoimmune mediated disease process which gives rise to
complete deficiency of insulin and so total dependency upon insulin for survival.

Type 2 diabetes:
Type 2 diabetes is generally occurring in adult, which may develop due to insulin
resistance and relative insulin deficiency or from a secretory defect. These diseases have a very
strong genetic predisposition and caused by a combination of inadequate insulin secretion and an
insensitivity of the body tissues to insulin.

Type 3 diabetes:
Type 3 diabetes; specific types of diabetes including various genetic defects in insulin action,
and diseases of the exocrine pancreas.

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Diagnosis:
Normal blood glucose level is 80-120 mg/dl. Due to insulin deficiency glucose level is
increased and it may 180-200 mg/dl which require immediate medical attention.
Diabetes mellitus is characterized by:
1. Hyperglycemia (Increase glucose level in blood)
2. Glycosuria (Presence of sugar in urine)
Symptoms of diabetes mellitus:
1. Polyphagia (Increased appetite)
2. Polydipsia (Increased thirst)
3. Polyuria (Increased urine output)
Blood sugar level in diabetes mellitus may increase due to following:
1. Increased absorption of glucose.
2. Decreased entry and oxidation of glucose in muscular tissue.
3. Decrease glycogen formation in liver
4. Increased glycogen breakdown in the liver.
Complication of diabetes mellitus:
 Neuropathy
 Cataract and retinopathy
 Nephropathy
Treatment of diabetes mellitus:
a) Intake of low carbohydrate and high protein diet.
b) Adequate exercise.
c) Use of oral antidiabeteic drugs in mild diabetes.
d) In severe case of diabetes use insulin.

II. Hypoglycemia:

Hypoglycemia, also known as low blood glucose, it occurs when blood glucose decrease
below 60 mg/dl. Glucose is an essential source of energy for the body, and obtained from food.
Carbohydrates are the chief source of glucose. Rice, potatoes, bread, tortillas, cereal, milk, fruit,
and sweets are rich source of carbohydrate.
After taking food, glucose is absorbed into the bloodstream and carried to the body’s
cells. Insulin hormone secreted from β-cells of pancreas and helps in the absorption of glucose to
obtain energy. If a person consumes more glucose than the body requirement, it stores extra
glucose in the liver and muscles in the form of glycogen. The body can use glycogen to obtain
energy. Extra glucose can be converted to fat and stored in fatty cells which can also use for
energy.
Hypoglycemia can appear suddenly. It is generally mild and can be treated easily by
eating or drinking a small amount of glucose-rich food. If left untreated, hypoglycemia can
become worse and leads to confusion, clumsiness, or fainting. Severe hypoglycemia may
develop seizures, coma, and even death.

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In adults and children of age than 10 years, hypoglycemia is rare except as a side effect of
diabetes treatment. Hypoglycemia can also result, however, from other medications or diseases,
hormone or enzyme deficiencies, or tumors.
Symptoms of hypoglycemia:
Following symptoms may occur in hypoglycemia
 Hunger
 Shakiness
 Nervousness
 Sweating
 Dizziness or light-headedness
 Sleepiness
 Confusion
 Difficulty speaking
 Anxiety
 Weakness
Hypoglycemia may occur in during sleep. Signs of hypoglycemia during sleep include
following:
 Crying out or having nightmares
 Finding pajamas or sheets damp from perspiration
 Feeling tired, irritable, or confused after waking up

Causes hypoglycemia in people with diabetes

Diabetes Medications
Hypoglycemia may occur as a side effect of some diabetes medications, including insulin and
oral antidiabeteic—pills— that increase insulin production, such as
 Chlorpropamide
 Glimepiride
 Glipizide
 Glyburide
 Nateglinide
 Repaglinide
 Sitagliptin
 Tolazamide
 Tolbutamide
Some combination pills may cause hypoglycemia, including
 Glipizide + metformin
 Glyburide + metformin
 Pioglitazone + glimepiride
 Rosiglitazone + glimepiride
 Sitagliptin + metformin

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Other diabetes pills, when taken alone, do not cause hypoglycemia. Examples of these
medications are
 Acarbose
 Metformin
 Miglitol
 Pioglitazone
 Rosiglitazone
Other causes of Hypoglycemia
In patient who are on insulin or pills that increase insulin production, low blood glucose can be
due to
 Meals or snacks that are too small, delayed, or skipped
 Increased physical activity
 Alcoholic beverages
Hypoglycemia can bet treated by giving sufficient glucose to the patient to restore glucose level.

III. Glycosuria:
Glycosuria may be defined as excretion of glucose in urine. Glomerular filtrate contains
glucose in same amount as blood plasma. This glucose reabsorbed in renal tubes and note appear
in urine in normal course. When blood glucose level increased, it is excreted in urine. This extra
glucose is not reabsorbed. So this passed in urine and leads to glycosuria.
Glycosuria can be classified in following type:
1. Hyperglycemic glycosuria
2. Alimentary glycosuria
3. Renal glycosuria

1. Hyperglycemic glycosuria:
Blood glucose level increased due to following:
Decrease insulin secretion
Increased activity of anterior pituitary, adrenal, thyroid hormones.
Excess glucose eliminated in urine and produce glycosuria.

2. Alimentary glycosuria:
This is a temporary condition and produced by taking high amount of carbohydrates, this
is rapidly absorbed, in some cases where a part of the stomach is surgically removed. This
excessive glucose excreted in urine produce glycosuria.

3. Renal glycosuria:
In this case blood glucose level does not increased but it occurs due to defective tubular
mechanism of glucose reabsorption. Hence glucose is not reabsorbed and appear in urine.

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