UNIVERSITI PERUBATAN ANTARABANGSA

INTERNATIONAL MEDICAL UNIVERSITY

MALAYSIA

Bachelor of Science (Honours)

Biomedical Science Programme

Endocrine System
BBS3012
(2 Credits)

Semester 5

2011

Laboratory Manual
Determination of glucose in body fluids (blood, urine and CSF)
Fasting blood glucose:
Fasting blood glucose is estimated 8 10 hours after the last meal. This is usually done in
the early morning. The person should not consume anything other than water.

Postprandial blood glucose:

Postprandial blood glucose is estimated usually two hours after a normal meal or
breakfast.

Random blood glucose:

The random blood glucose can be measured at anytime of the day irrespective of food
taken.

There are many methods available to measure the blood glucose level. They are.
1. Enzymatic method
a) Hexokinase method
b) Glucose oxidase peroxidase method (GOD POD)
2. Polarographic method
a) PO2 electrode method
3. Chemical methods
a) O toluidine method
b) Folin Wus method
c) Nelson somogyis method

Glucose Oxidase Peroxidase method

Principle:

The enzyme,glucose oxidase (GOD) catalyses the oxidation of glucose to gluconic acid
and H2O2. Addition of the enzyme peroxidase splits H2O2 to H2O and nascent oxygen.
The oxygen oxidizes the added colourless chromogen 4 Amino antipyrine to a coloured
compound which can be read colorimetrically. The intensity of the colour is directly
proportional to the glucose concentration. The method is highly specific and gives an
accurate glucose value. The glucose oxidase act only on D- Glucose.
Glucose Oxidase
-D-Glucose + H2O + O2 ------------------------> H2O2 + D-Gluconic Acid

Peroxidase
H2O2 + 4-Aminoantipyrine + Phenol ------------------> Iminoquinone + H2O

Reagents:

1. Buffer solution: 0.2M phosphate buffer, pH 7.4.

Solution A: 0.2 M monobasic sodium dihydrogen phosphate (NaH2PO4.2H2O) is

prepared by dissolving7.8g in 250ml water.

Solution B: 0.2M dibasic disodium hydrogen phosphate ((Na2HPO4.2H2O) is

prepared by dissolving 8.9gm in 250ml water.

The two solutions are mixed as follows and the pH is adjusted to 7.4.
36.4ml of solution A + 213.6ml of solution B.

2. Phenol: 0.05%, 100mg 0f phenol taken directly.

3. Color reagents:

a. 4 Amino antipyrine
b. Horse radish peroxidsae: 0.73IU/ml 1mg/100ml of buffer
c. Glucose oxidase 30IU/ml 22mg/100ml of buffer.
d. Mix a + b+ c. refrigerate (do not freeze). To this add 100mg of phenol.

4. Glucose standard (stock): 1g of glucose is dissolved in 100 ml water. For

storage 100mg of benzoic acid(0.1% benzoic acid) + 100ml water + 1gm of
glucose

Working standard preparation:

a. 50mg/dl 0.5ml stock + 9.5 ml of distilled water

b. 100mg/dl 1ml stock + 9ml of distilled water
c. 150mg/dl 1.5ml stock + 8.5of distilled water
d. 200mg/dl 2ml stock + 8ml of distilled water
e. 250mg/dl 2.5ml stock + 7.5ml of distilled water
f. 300mg/dl 3ml stock + 7ml of distilled water
g. 350mg/dl 3.5ml stock + 6.5ml of distilled water
h. 400mg/dl 4ml stock + 6ml of distilled water.
 Procedure

Standard glucose (0.02ml of each) are pipetted out into S1 S8 series of test tubes.
Simultaneously run the blank of 0.02ml of water. Serum of 0.02ml pipetted into a test
tube labeled test. Then 1ml of water is added to all tubes, mixed well and 2ml of
colour reagent are added. Contents are mixed and incubated at 370C for 15 minutes.
The absorbance of the red colour developed is read at 505nm. Colour is stable for 1
hour at room temperature.

Contents B S1 S2 S3 S4 S5 S6 S7 S8 T
Working - 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 -
standard (ml)
Conc.mg/dl - 50 100 150 200 250 300 350 400 -
Serum (ml) - - - - - - - - - 0.02

Water(ml) 1.02 1 1 1 1 1 1 1 1 1
Colour
reagent (ml) 2 2 2 2 2 2 2 2 2 2

Mix and incubate at 370C for 15min or at room temperature for 30 minutes and
read at 505nm.

Calculations:

Glucose concentration (mg/dl) = Absorbance of test / Absorbance of standard x 100

Calculate the glucose concentration also using the standard curve.

To convert mg/dl to mmol/L

mmol/L = mg/dl x 0.056
Oral glucose tolerance test:

A normal person should be able to reduce a glucose load from his blood within a
specified time. This is known as normal tolerance.

If the person has an elevated blood glucose concentration for longer than the normal time,
the condition is called reduced tolerance.

If the glucose concentration becomes very low or normal very early than the normal time
then the condition is called as increased tolerance.

The tests that are used to measure these changes in blood glucose after glucose load are
called glucose tolerance tests. Oral glucose tolerance is more commonly used in all
laboratories.

Indications of glucose tolerance test:

1. From the family history of diabetes mellitus
2. Signs and symptoms comparable with diabetics without any
complications
3. Border line glucose In PPBS (Postprandial Blood Sugar)
4. Reactive hypoglycaemia for 3hrs or longer period after food intake.
5. Pregnancy of history of abortions, still births or a large baby.

Preparations of the patients

1. Patient should not be under fear or anxiety about the possibility of being diabetic.
If so it can lead to false positive results. So it is the duty of the technician to
prepare the patient emotionally and mentally calm.
2. Adequate carbohydrate intake: Before the test, the patient should have been on a
diet containing at least 150g of carbohydrate per day with low fat for at least
3days.
3. It is desirable for the subject to fast 8 10hr before the test.
4. The patient must not have ingested tea or coffee on the day of test.
5. The patient should not have excessively exercise or physically strained.
6. If the patient is not well the test should be postponed
 7. The patient should not receive any drugs for at least 3days before the test.

Method:
The test is usually carried out in the early morning after fasting overnight.
Fasting blood sample and urine is then collected. 75g (or100g) of glucose dissolved in
about 150 200ml of water is given to drink.

Venous blood for the estimation of blood glucose is collected at 30 min intervals for 2
21/2 hrs or hourly intervals for 3hours after the ingestion of glucose. Urine specimen is
also collected at the same time. Blood glucose in each sample and the urine is tested for
the presence of sugar.

Example for normal glucose tolerance curve:

Sample No. mg glucose/100ml blood Urine glucose

Fasting 90 Negative
30min 120 Negative
60min 150 Negative
90min 140 Negative
120min 90 Negative

Example for abnormal glucose tolerance curve:

Sample No. mg glucose/100ml blood Urine glucose

Fasting 120 Negative
30min 160 Negative
60min 200 Negative
90min 180 Negative
120min 150 Negative
Determination of serum Calcium (Trinder Method)

Principle:
Calcium is precipitated with naphthyl hydroxamic acid. The precipitate is dissolved in
EDTA and the colour is developed with ferric nitrate. The absorbance of colour
compound is measured in colorimeter and the calcium level is determind.

Reagents:
Calcium reagent:
In a 250ml beaker add 100ml water and 5ml ethanolamine. Add 2g of tartaric acid and
Mix. Add 250mg of napthyl hydroxamic acid and dissolve by warming. In a litre flask
add 9g sodium chloride and 500ml of water. Pour the contents of the beaker into the 1L
flask containing sodium choloride solution. Add water upto 1L mark. Mix. Filter through
whatman No. 40 filter paper. The reagent is stable for months.

EDTA solution:
Dissolve 2g of disodium ethylene diamine tetra acetate in 1L of 0.1N NaOH.
Colour reagent:
Dissolve 60g of ferric nitrate, in 500ml of water, add 15 ml of concentrated nitric acid
and add water up to 1litre.

Calcium standard, 5mEq/L:

Dissole 125mg of dry calcium carbonate (dried at 1200C for 30 minutes in a hot air oven)
in 40ml of 0.1 N hydrochloric acid and dilute to 500ml with water.

Procedure:

Reagents B S T
Serum - - 0.2ml
Standard calcium - 0.2ml -
Calcium reagent 5.0ml 5.0ml 5.0ml

Mix .stand 30 minutes at room temperature. Centrifuge. Pour off

supernatant
EDTA solution 1.0ml 1.0ml 1.0ml
Keep in boiling water bath, 10 minutes, cool
Colour reagent 3.0ml 3.0ml 3.0ml
Read the absorbance at 450nm or blue filter

OD of T OD of B
Serum Calcium in mEq/L =
OD of S OD of B

To convert the result to mg% multiply mEq/L by 2.

Determination of Serum Inorganic Phosphorus (Fiske Subba Row
method)

Specimen: Serum collected from venous blood. Plasma is also suitable.

Principle:
Serum proteins are precipitated by trichloroacetic acid. Molybdic acid is added to protein
free filtrate converts phosphate to phosphomolybdate. Alpha napthol sulfonic acid
added reduces phosphomolybdate to blue coloured compound. The intensity of blue
colour is measured photometrically using red filter or at 660nm.

Reagents:
1. Trichloroacetic acid, 10% . Dissolve 10g TCA in water and make up to 100L.
2. Molybdate reagent: To 200ml of water add 83ml sulfuric acid, keeping cold
under tap or cold water. Dissolve 25g ammonium molybdate in this soloution.
Dilute to 1 litre with water. Solution is stable.
3. 1,2,4 Amino napthol sulfonic acid reagent(ANSA) : Dissolve 0.125g of
1,2,4 - amino napthol sulfonic acid, 7.28g of sodium metabisulfite and 0.25g of
anhydrous sodium sulfite in 50ml of water. Filter, store in a brown bottle. The
agent is stable for one month kept in refrigerator.
4. Standard phosphorous solution: 0.08mg per ml solution.
Dissolve 0.351g of dried potassium hydrogen phosphate in about 500ml water in
a litre flask. Add 8ml concentrated HCl and dilute to1litre. Prepare fresh once a
month.
Procedure:

Reagents Blank (B) Standard (S) Test (T)

Serum - - 1ml
TCA, 10% - - 9ml
Mix. Stand for 5 minutes, filter
Water (ml) 1 0.5 -
Standard
phosphorus solution - 0.5 -
(ml)
- - 5
Filtrate (ml)
4 4 -
TCA,10% (ml)
Molybdate reagent
(ml) 1 1 1
ANSA 0.4 0.4 0.4

Mix. Stand for 5minutes

Water (ml) 3.6 3.6 3.6
Read at 660nm or red filter

Calculations:
100ml serum contains = T B/S B x 4
T = Absorbance of test
B = absorbance of Blank
S = Absorbance of Standard