Glucose estimation

ESTIMATIONOF GLUCOSE
Dr. Azad Alam Siddiqui
Assistant Professor
Department of Vocational studies (MMDT)
KMGGPGC, Badalpur, GB Nagar, UP

OBJECTIVES
 To know the different methods for estimation of blood
glucose
 To know the precautions needed to get accurate results
and better interpretation of glycemic status in relation
to disease condition.

INTRODUCTION
 Glucose is a simple sugar which is a permanent and immediate primary source
of energy to all of the cells in our body. Body tries to maintain a constant supply
of glucose for your cells by maintaining a constant blood glucose concentration.
The concentration of glucose in blood, expressed in mg/dl, is defined by the
term glycaemia. Blood sugar levels are regulated by the hormones insulin and
glucagon which act antagonistically. These two hormones are secreted by the
islet cells of the pancreas. Essentially blood glucose levels determine the time of
secretion of these hormones.
 The blood glucose level is easily changed under the influence of some external
and internal factors such as body composition, age, physical activity and sex.
Diabetes is a disease related by the abnormal metabolism of blood sugar and
defective insulin production. So blood sugar levels are an important parameter
for the study of diabetes. The level of glucose circulating in blood at a given time
is called as blood glucose level. The blood glucose level varies at different time
on various part of the day.

INTRODUCTION CONT….
 Hypoglycaemia is a possible side effect of diabetes medications in which
blood glucose level drops below 70mg/dl. Hypoglycaemia is less
frequently observed, but is found in conditions such as insulinoma,
hypopituitarism, neoplasms, or insulin-induced hypoglycaemia.
 In people with diabetes, the body doesn't produce enough insulin or
respond to insulin properly. The result is that sugar builds up in the
blood stream, damaging the body's organs, blood vessels and nerves.
This condition in which too much sugar in the blood stream is called
hyperglycaemia.
 The most frequent cause of hyperglycaemia is diabetes mellitus. Some
other factors that contribute to elevated blood glucose are pancreatitis,
pituitary or thyroid dysfunction, renal failure, and liver disease.
 Arterial blood has more glucose than venous blood (because the tissues
have typically used some glucose).

ENTRY OF GLUCOSE INTO THE CELL
• Two specific transport system are used :
• 1. Insulin –independent transport system:
• • Carrier mediated uptake of glucose
• • Not dependent on insulin.
• • Present in hepatocytes, erythrocytes & brain.
• 2. Insulin dependent transport system :
• • Present in Skeletal muscle.

• For glucose estimation from any material, blood is
collect in fluoride containing vial.
• Fluoride inhibit glycolysis by inhibiting enolase
enzyme.
• In CSF, bacteria & other cells are also present so
analysed immediately.
• For glucose estimation from urine, add 5ml glacial
acetic acid as preservative to inhibit bacterial
growth.

 The glucose can be measured in whole blood or separated blood
(plasma/serum). Whole blood has a lower glucose level because the red cells
don't have much glucose in them. If the serum / plasma is not separated from
the cells immediately, the blood cells (which stay alive for many hours in vitro)
will use up some of the glucose and usually generate lactate. The other ways to
stop the glucose falling in a plasma or serum sample are putting the sample on
ice or adding a glycolysis inhibitor such as fluoride or citric acid, or both.
 The term blood sugar often includes, besides glucose, other hexoses as well as
other reducing substances present in the blood.
 There are two methods to estimate blood glucose:
1. Glucose oxidase method
2. Methods depends on reducing property of glucose

FUNCTION
 ENERGY SOURCE
• Glucose is a ubiquitous fuel in biology, providing approximately 3.75
kilocalories (16 kilojoules) of food energy per gram. Breakdown of
carbohydrates (e.g. starch) yields mono- and disaccharides, most of which is
glucose.
• Glucose is a primary source of energy for the brain, and hence its
availability influences psychological processes. When glucose is low,
psychological processes requiring mental effort (e.g., self-control,
effortful decision- making) are impaired.
 NORMAL VALUES
Category of a person Fasting Value Post Prandial
Minimum Value Maximum Value Value 2 hours after
consuming glucose
Normal 70 100 Less than 140
Early Diabetes 101 140 140 to 200
Established Diabetes More than 140 - More than 200

1. GLUCOSE HEXOKINASE METHOD
• The enzyme hexokinase (HK) catalyses the reaction between
glucose and adenosine triphosphate (ATP) to form glucose-6-
phosphate (G-6-P) and adenosine diphosphate (ADP). In the
presence of nicotinamide adenine dinucleotide (NAD), G-6-P is
oxidized by the enzyme glucose-6-phosphate dehydrogenase (G-6-
PD) to 6-phosphogluconate and reduced nicotinamide adenine
dinucleotide (NADH). The increase in NADH concentration is
directly proportional to the glucose concentration and can be
measured spectrophotometrically at 340 nm.
Glucose + ATP + HK  ADP + G6P
G6P + NAD + G6PD  6 P-gluconolactone
+NADH+H

2. ALKALINE COPPER REDUCTION METHOD (FOLIN
AND WU METHOD)
 This method employed for the estimation of blood glucose
because of the ability of glucose to reduce alkaline copper
solutions. Reagents containing phosphomolybdic acid are
frequently used to form a blue complex by combination with the
reduced copper.
 When glucose or other reducing agents are treated with alkaline
copper solution they reduce the copper with the result insoluble
cuprous oxide is formed. The reaction depends on temperature,
duration of heating, degree of alkalinity. The cuprous oxide form
is allowed to react with phosphomolybdate to form molybdenum
blue coloured complex which can be read calorimetrically using
red filter on at 680nm.

3. OXIDASE PEROXIDASE METHOD
 Glucose oxidase is an enzyme extracted from the growth medium of
Aspergillus niger. Glucose oxidase catalyse the oxidation of β D- glucose
present in the plasma to D glucono -1,5 - lactone with the formation of
hydrogen peroxide; the lactone is then slowly hydrolysed to D-gluconic
acid. (Glucose oxidase catalyses the aerobic dehydrogenation of β-D-
glucose, thus forming gluconic acid and hydrogen peroxide.) The hydrogen
peroxide produced is then broken down to oxygen and water by a
peroxidase enzyme. Oxygen then react with an oxygen acceptor such as o-
toluidine or 4-aminoantipyrine which itself converted to a coloured
compound, the amount of which can be measured colorimetrically.
Glucose Oxidase
 Glucose + O2 +H2O Gluconic acid +H2O2
Peroxidase
• H2O2 + Phenol + 4-aminoantipyrine Quinoneimine +H2O

Requirements :
*Samples:
 Blood samples
--Whole blood
--Serum
--Plasma (with Ca.oxalates / NaF), which is the preferred
sample
Instrumentation:
-Photometer adjusted on wavelength 505 nm
- Cuvette (light path) 1 cm
-Water bath at 37 ºC
-Automatic pipettes, disposable test tubes , racks and disposable
tips for the dispensers.

PROTOCOL
• Method
 The test tubes were marked T1 to T6, for those that had to be
inoculated with glucose. They were arranged in order of concentration
on the rack.
 Inoculation commenced by transferring glucose into different test
tubes T1 being the least concentrated. 0.5mM of Glucose was
transferred using a pipette. 0.2ml was inoculated into T2, 0.4 ml into
T3, 0.6 ml into T4, 0.8ml into T5 and 1.0 ml into T6.
 Distilled water was then inoculated into the test tubes using a
different pipette to avoid cross contamination. 1ml was inoculated
into T1, 0.8 ml into T2, 0.6 ml into T3, 0.4 ml into T4 and 0.2 ml into
T5. There was no water inoculated into the last tube T6.

PROTOCOL CONT…
 Phenol (0.5ml) was then inoculated into all the test tubes. It was
transferred using a different pipette to avoid cross contamination.
 1.5ml of GOD-POD reagent was then inoculated into all the test tubes
using an automatic pipette and a long pipette tip. The test tubes were
then agitated on the rack and incubated in the water bath for (15 -30)
minutes at 370C. The temperature was constantly checked during
incubation.
 After (15-30) minutes, the solutions changed colour from colourless to
light pink according to the concentration. These different solutions
were then read on a spectrophotometer in a cuvette. The
spectrophotometer was zeroed at first then absorbencies of glucose
was read and recorded.
 A cuvette was wiped on the soft side to minimize absorbencies caused
by contamination. These different absorbencies were recorded on a
table.

PROCEDURE
• Mix and incubate for 15 minutes at 370C. Mix and read
absorbance of standard(S) and test against blank(B) at
505nm. The final colour is stable for 10 hr at R.T.
Reagent For 1ml procedure
B S T
Glucose working
Reagent
1.O ml 1.O ml 1.O ml
Glucose Standard
(conc.100mg/dl)
----------------------- 10µl ------------------------
Sample ----------------------- ------------------------- 10µl

CALCULATIONS
•Glucose Conc. (mg/dl) =
•Abs. of T/Abs. of S × Conc. of
standard Glucose

GLUCOSE ESTIMATION IN CSF
• CSF is a fluid that flows through and protects the subarachnoid
space of the brain and spinal cord.
• It is obtained by lumbar puncture, L 3-L 4.
• In CSF, Glucose is estimated by GOD – POD method.
• CSF Contain-
• 15– 45 mg% Glucose

GLUCOSE ESTIMATION IN CSF
• Clinical interpretation:
• An increased CSF glucose level is seen in hyperglycaemia.
• Decreased CSF glucose in
1. Bacterial Infection
2. Hypoglycaemia

CLINICAL SIGNIFICANCE OF GLUCOSE ESTIMATION
 Increased glucose : (hyperglycaemia)
• Diabetes mellitus,
• Hyperthyroidism,
• Hyperpituitarism,
• Adrenocortical hyper activity,
 Decreased glucose: (hypoglycaemia)
• Hypothyroidism,
• Hypopituitarism,
• Hypoadrenalism,

CONCLUSION
 The Folin Wu method though sensitive is nonlinear. The fading of colour
during reading of absorbance adds to this imprecision and inaccuracy.
Folin Wu method require protein precipitation, centrifugation and
boiling which is time consuming. Folin Wu method requires special tubes
(Folin Wu tubes) and accurate boiling time (6 minutes). The absorbance
should be read immediately as colour fades rapidly or at fixed interval
(such as 5 minutes) which is technically not feasible.
 The GOD-POD method is linear (up to 500 mg/dl), sensitive (detection
limit 0.3 mg/dl), simple (requires 10 microlitre of sample to be
incubated for 15 minutes with single reagent at 370C) and requires
simple instrumentation (the absorbance to be read between 505 nm to
550 nm). When the results obtained by this method using Indian kit, is
compared with that of Hexokinase method using a kit from Sigma
Chemical Co. USA, the average deviation was found to be -0.97 per cent
only. Thus the GOD-POD method which is comparatively cheap, the kit
for which is readily available and can be adopted using colorimeter, is
best method for glucose estimation.

Glucose estimation

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Glucose estimation