PRACTICAL BIOCHEMISTRY

GDN202
PRACTICAL ANALYSIS USING SPECTROPHOTOMETER

In many diseases we need to measure the

concentration of many substances of
medical interest in biological blood e.g.
glucose in diabetes mellitus, uric acid in gout
CLINICAL BIOCHEMISTRY

Blood tests
Blood glucose

Blood Lipid Profile

Liver Functions

Kidney Functions
 Colorimetric
estimation of
blood glucose
ILO OF THE CURRENT TOPIC:

By the end of this topic, the student will be able to:

1) Explain the medical importance of determination of
plasma glucose concentration.
2) Explain the principle of estimation of blood glucose
concentration by
glucose oxidase reaction .
3) Perform the steps of the experiment with accurate
pipetting of the volumes required in the experiment.
4) Interpret the resulting data whether it shows
normoglycemic or hyperglycemic or hypoglycemic results.
5) Correlate the results to possible medical problem
causing such changes in plasma glucose level.
 Blood = Fluid tissue

Extracellular Specialized cells:

matrix: (= Formed elements)
Plasma (55%) RBCs

WBCs

Platelets
SPECIALIZED CELLS:

Red blood cells

White blood cells

Platlets
PLASMA COMPOSITION

Transports organic and

inorganic molecules,
formed elements, and heat

Water 90%
Plasma proteins
Other solutes
BLOOD SAMPLE PROCESSING
Difference between
plasma and serum?
serum = plasma clotting factors
 PLASMA AND SERUM
Differences Plasma Serum
Blood is allowed to
Blood is mixed with
clot at room
anticoagulant
Preparations temperature for 15`-30`
centrifugation The
centrifugation The
supernatant fluid is then
supernatant fluid is then
separated.
separated.
Fibrinogen &
Present Absent
Prothrombin
More turbid Is clearer
Appearance
(more protein) (fewer proteins)
Plasma less preferred
Preference in clinical Serum is the most
(the interferences by
chemistry preferred.
anticoagulants.
BLOOD COMPOSITION
USES OF BLOOD TESTS

Assessing organs

Checking for infection

Medical importance of blood glucose
determination

Blood glucose determination

Diagnosis and care of diabetic

patients
- Blood glucose testing allows for
quick response to high blood sugar
(hyperglycemia) or low blood sugar
(hypoglycemia).
 Causes of hyperglycemia

1-Diabetes mellitus : Type 1 & type 2.

2- High epinephrine : severe emotional
stress.
3- Gestational diabetes.
 Causes of hypoglycemia

a) Over dose of insulin or oral

hypoglycemic drugs.
b)Insulinoma.
c) Chronic alcoholism
colorimetric estimation of blood
glucose
by glucose oxidase reaction
 Principle of the glucose oxidase reaction

Glucose is oxidized by glucose oxidase to form gluconic

acid with liberation of hydrogen peroxide.

Glucose + O2 + H2O glucose oxidase

Gluconic acid + H2O2

 Principle of the glucose oxidase reaction

Hydrogen peroxide is dissociated to water and oxygen

atom by peroxidase enzyme.
The liberated oxygen is captured by a chromogen
( a mixture of 4-amino antipyrine & phenol) which is
converted to a red colored product.

H2O2 + Phenol + 4-amino antipyrine peroxidase

red colored product
 Materials required
Clean dry pipettes & test tubes.
A pump.
Reagents
Known glucose solution (Standard)
Unknown glucose solution
Distilled water
Step 1:Label 3 dry test tubes

S B
T

For the test For the For the

sample Standard Blank
sample
 Step 2: Practical

Determine the glucose concentration in the provided sample as

follows:

Blank Standard Sample

Distilled 0.5ml ___ ___
water
Glucose ___ 0.5ml ___

standard
Serum/pla ___ ___ 0.5ml
sma
glucose
1 ml 1 ml 1 ml
reagent
Step 3: for all ; Pipette 1ml of the
glucose reagent into the previous 3
test tubes.
Step 4: Pipette 0.5ml of the given Test
sample-Standard-DW into their corresponding
dry test tubes
o o o

0.5 0.5 0.5

ml ml ml

0.9 0.9 0.9

 T S B

Step 5:Mix the contents of the tubes

and let stand for 5 minutes at room
temperature.
Step 6:Record the absorbance of both
test & standard against the blank at
520 nm.
 Calculate the test concentration by the
following equation:

Concentration of blood glucose ( mg/dl) =

Absorbance of Test
----------------------- X Concentration of standard
Absorbance of standard

Remember
the check List
Students will
be divided
into groups

Students will
examine urine
sample

Students will
estimate blood
glucose levels in the
provided samples

Write final
comment
 MCQ

A fasting blood glucose test level of

_________________ indicates
prediabetes.

A. 50 mg/dl to 69 mg/dl
B. 70 mg/dl to 99 mg/dl
C. 100 mg/dl to 125 mg/dl
D. 126 mg/dl or higher on two separate
tests
 WHICH OF THE FOLLOWING CONFIRMED VALUES
MEET THE DIAGNOSTIC THRESHOLD FOR
DIABETES?
A. fasting blood glucose equal to or greater
than 140mg/dl
B. random glucose greater than 160 mg/dl
C. 2 hour post prandial glucose greater than
or equal to 126 mg/dl
D. fasting blood glucose equal to or greater
than 126mg/dl
Which endocrine imbalance would cause
hypoglycemia?
A) hypersecretion of insulin
B) hypersecretion of glucagon
C) hypersecretion of cortisol
D) hypersecretion of growth hormone
CASE 1

A 45-year-old obese man presented with excessive

thirst, frequent urination and blurring of vision . He
has lost 5 pounds in the past month instead of
increased food intake. At the doctor's office, blood and
urine samples were taken.
Perform complete urine report and comment on the
results.
Perform colorimetric estimation of blood glucose and
comment on the results