Download Estimates of The Glucose Concentration

Lab 4
M. Zaharna Clin. Chem. Lab
All body tissues can utilize glucose, the principle
and almost exclusive carbohydrate circulating in
blood.
 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

M. Zaharna Clin. Chem. Lab
 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);
Those associated with decreased plasma glucose
concentration (hypoglycemia);
M. Zaharna Clin. Chem. Lab
 Estimates
of the glucose concentration in
blood are required to:





help in the diagnosis of diabetes mellitus
management of DM patients
and monitoring of treatment in DM patients
It is also essential for the detection and proper
management of hypoglycemia, a condition
encountered much less frequently
 neonatal hypoglycemia
or any other condition in which there is
abnormal carbohydrate metabolism in the
body.
M. Zaharna Clin. Chem. Lab
 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 orthotoluidine test and later by enzyme
methods employing either glucose oxidase
or hexokinase.
M. Zaharna Clin. Chem. Lab
 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.
M. Zaharna Clin. Chem. Lab
 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: 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
M. Zaharna Clin. Chem. Lab
 Method
3: 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. (Read Absorbance at 340nm)

 Method
4: Glucose oxidase coupled reaction,
Quantitative

One of the most frequently used specific glucose
methods uses two coupled enzyme reactions
 Method
5: Glucose dehydrogenase;
quantitative,

Increased absorbance at 340 nm is related to glucose
concentration (NADH produced)
M. Zaharna Clin. Chem. Lab
 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.
M. Zaharna Clin. Chem. Lab
 The
addition of 2 mg sodium fluoride per
mL blood to be collected will prevent
glycolysis for 24 hours.
• sodium fluoride
Potassium oxalate/
• Sodium fluoride/
Na2 EDTA
• Sodium fluoride
(serum tube)
M. Zaharna Clin. Chem. Lab
• For glucose determinations
Oxalate and EDTA
anticoagulants will give
plasma samples. Sodium
fluoride is the
• antiglycolytic agent.
• Tube inversions ensure
proper mixing of additive
with blood.
 Serum,
plasma is suitable for samples.
 Whole blood and hemolysis are not
recommended for use as a sample.
 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
M. Zaharna Clin. Chem. Lab
M. Zaharna Clin. Chem. Lab
 Diabetes
mellitus, as previously stated is a
condition of hyperglycemia.
 It is estimated that this condition affects 2.55% 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.
M. Zaharna Clin. Chem. Lab
 There
is a relationship between control of
the glucose concentration fluctuation and
the progression of the disease
complications.
 There should be a method to quantify
accurately and objectively the degree of
altered blood glucose control over a long
period of time.
M. Zaharna Clin. Chem. Lab
In adults, hemoglobin is a mixture of three
forms: Hb A1, Hb A2, and Hb F, with Hb A1
predominating.
 Hemoglobin A1 consists of three subforms: Hb
A1a, Hb A1b, and Hb A1c, with Hb A1c
predominating.
 The term glycated hemoglobin describes a
chemically stable conjugate of any of the forms
of hemoglobin with glucose.
 Glycated forms of hemoglobin are formed slowly,
nonenzymatically, and irreversibly at a rate that
is proportional to the concentration of glucose in
the blood.

M. Zaharna Clin. Chem. Lab
 As
blood glucose levels rise, the increase in
glycosylation of proteins is proportional to
both the level of glucose and the lifespan of
the protein being affected.
 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 period (90-120 days) .

M. Zaharna Clin. Chem. Lab
 Today,
hemoglobin A1C (HbA1c) testing is
performed to monitor diabetics suspected
of having irregular control over their
glucose level.
 Glycated hemoglobin measurements are,
however, influenced by conditions that
affect the life span of the hemoglobin
molecule, such as sickle cell disease and
hemolytic disease, which can falsely
decrease glycated hemoglobin results.
M. Zaharna Clin. Chem. Lab
 By
testing for glycosylated hemoglobin, the
doctor discovers what the average blood
glucose level has been for the previous 2 to
3 months.
 This is especially valuable when monitoring
diabetics whose blood sugars change
dramatically from day-to-day and to monitor
long-term diabetic control.
 Whereas a fasting blood sugar may be
influenced by the patient’s recent adherence
to the prescribed treatment regimen,
M. Zaharna Clin. Chem. Lab
 The
glycosylated hemoglobin is
irreversible; it shows what type of
diabetic control has occurred over several
months.
 If the diabetes is controlled (basically an
HbA1C lower than 7%), the test should be
done every 3-6 months.
M. Zaharna Clin. Chem. Lab
M. Zaharna Clin. Chem. Lab
 EDTA
is the anticoagulant of choice for all
methods.
 No special preparation, fasting specimens are
not required
 Most methods require cell lysis with a
hemolyzing reagent provided by the
manufacturer prior to loading.
 Typically, whole blood may be stored up to 7
days at 2–8° C.
M. Zaharna Clin. Chem. Lab


Nondiabetic adult: 3-5 %
Diabetic adult: <7%
Avg. Blood Sugar
HbA1c
(%)
(mmol/L)
(mg/dL)
4
3.3
60
5
5.0
90
6
6.7
120
7
8.3
150
8
10.0
180
9
11.7
210
10
13.3
240
11
15.0
270
12
16.7
300
13
18.3
330
14
20.0
360
M. Zaharna Clin. Chem. Lab