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Introduction
• All body tissues can utilize glucose, the principle and almost
exclusive carbohydrate circulating in blood.
• Glucose is a reducing monosaccharide that serves as the
principal fuel of all the tissues. It enters the cell through the
influence of insulin and undergoes a series of chemical
reactions to produce energy.
• The glucose level in the blood is maintained within a narrow
range under diverse conditions by regulatory hormones
• Measurement of glucose is one of the most commonly
performed procedure in most hospital chemistry laboratories
• The most frequently encountered disorder of carbohydrates
metabolism is a high blood glucose due to DM
Mohammed Laqqan
• The various disorders in carbohydrate metabolism may be
grouped into several categories dependent primarily upon
laboratory findings:
– those associated with a raised plasma glucose concentration
(hyperglycemia);
– a decreased plasma glucose concentration (hypoglycemia).
• Estimates of the glucose concentration in blood are required to
help in the diagnosis of diabetes mellitus or any other
condition in which there is abnormal carbohydrate metabolism
in the body.
• Lack of insulin or resistance to its action at the cellular level
causes diabetes .
Mohammed Laqqan
Mohammed Laqqan
Purposes of Determination
• Methods for the quantitative measurement of glucose
in blood were introduced at the beginning of the
century.
• The principle use of the test is for:
– the diagnosis and management of diabetes,
– but it is absolutely essential for the detection and
proper management of hypoglycemia, a condition
encountered much less frequently.
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• Measuring blood glucose is used for the diagnosis of:
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Carbohydrate metabolism disorders
Monitoring of treatment in diabetes mellitus
Neonatal hypoglycemia,
Idiopathic hypoglycemia,
Insulinoma.
Mohammed Laqqan
Methods of Analysis
• Until 15 or 20 years ago, the majority of the quantitative tests
for glucose determination depended upon the oxidation of
glucose by hot, alkaline copper solutions or solutions of
potassium ferricyanide.
• These were replaced by the ortho-toluidine test and later by
enzyme methods employing either glucose oxidase or
hexokinase.
• Enzymatic methods give a “true” glucose determination
because of the high specificity of an enzyme for a particular
substrate.
• There should be fewer interfering substances when measuring
the glucose concentration in serum using enzyme methods.
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• Regardless which method of glucose determination is
employed, precautions in the sample collection are
required to prevent the utilization of glucose by cells.
• The glucose loss, upon standing in a warm room, may
be as high as 10 mg/dL per hour.
• The decrease in serum glucose concentration is
negligible if the blood sample is kept cool and the
serum separated from the clot within 0.5 hours of
drawing.
• The addition of 2 mg sodium fluoride per mL blood
to be collected will prevent glycolysis for 24 hours.
Mohammed Laqqan
Methods of glucose analysis
• Method 1: Benedict’s: (qualitative, semiquantitative)
– This procedure, sensitive to total reducing compounds
present in urine, yields red Cu2O and yellow CuOH
precipitates.
• Method 2: Alkaline ferricyanide (quantitative)
– involves the reduction of yellow ferricyanide, Fe(CN)6, to
colorless ferrocyanide, Fe(CN)6, by glucose in alkaline
conditions.
• Method 3: o-Toluidine (quantitative)
– The o-toluidine reaction is based on the ability of many
aromatic amines in acid solutions to condense with the
aldehyde group of glucose to form glycosamines.
– Increased absorbance at 630 nm
Mohammed Laqqan
Enzymatic Methods
• Method 4: Hexokinase (HK) (quantitative)
– Involves two coupled reactions and two enzymes.
– Yield 1 mol of NADH or NADPH for each mole of glucose
that is oxidized. (340nm)
• Method 5: Glucose oxidase coupled reaction, (quantitative)
– One of the most frequently used specific glucose methods
uses two coupled enzyme reactions
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Glucose Oxidase Method
• Glucose present in the plasma is oxidized by the
enzyme glucose oxidase (GOD) to gluconic acid with
the liberation of hydrogen peroxide, which is
converted to water and oxygen by the enzyme
peroxidase (POD).
• 4 aminophenazone, an oxygen acceptor, takes up the
oxygen and together with phenol forms a pink
coloured chromogen.
Mohammed Laqqan
Mohammed Laqqan
Specimen collection and preparation
• Serum, plasma is suitable for samples.
• Freshly drawn serum is the preferred specimen.
• Stability: Serum, heparin or EDTA-plasma (with
addition of glycolytic inhibitor) :
– 2 days at 20–25oC
– 7 days at 2–8oC
• Note: Whole blood and hemolysis are not
recommended for use as a sample.
• Fluoride and iodoacetate have been used as inhibitors
of glycolysis to preserve blood that cannot be
separated rapidly.
Mohammed Laqqan
Mohammed Laqqan
Glycated Hemoglobin (HbA1C)
• Diabetes mellitus, as previously stated is a condition
of hyperglycemia.
• It is estimated this condition affects 2.5-5% of the
population and is considered to be the fifth leading
cause of death in the U.S.
• The disease is associated with a number of serious
micro and macro-vascular complications involving
the eyes, kidneys, heart and blood vessels, and may
greatly impair the quality of life or shorten the lifespan of the person afflicted.
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• Glycated hemoglobin is a form of hemoglobin which
is measured primarily to identify the average plasma
glucose concentration over prolonged periods of time.
• It is formed in a non-enzymatic glycation pathway by
hemoglobin's exposure to plasma glucose.
• Normal levels of glucose produce a normal amount of
glycated hemoglobin.
• As the average amount of plasma glucose increases,
the fraction of glycated hemoglobin increases in a
predictable way.
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• With consideration of RBC's lifespan, glycosylated hemoglobin
has been accepted as a measurement which reflects, better than
FBS determinations, the mean daily blood glucose concentration
and the degree of carbohydrate imbalance over the preceding
two months.
• Today, hemoglobin A1C testing is performed to monitor
diabetics suspected of having irregular control over their glucose
level.
• There are a number of techniques used to measure A1C.
Laboratories use:
 high-performance liquid chromatography
 immunoassay
Mohammed Laqqan
Mohammed Laqqan
Normal results
• An HbA1c of 6% or less is normal.
• If your HbA1c is above 6.5% you may be diagnosed
with diabetes.
• If you have diabetes, try to keep your HbA1c level at
or below 7%. However, you and your health care
provider must decide what a normal HbA1c level is
for you.
Mohammed Laqqan