February 11,

DETECTION OF DIABETES USING CHROMATOGRAPHY TECHNIQUE

2010

What is Diebetes?

Glucose is an important source of energy for the body. We obtain glucose either from the
diet or from production by the liver. Liver stored or released glucose to maintain the
optimum level of glucose in the blood. Insulin, a hormone synthesized by the pancreas,
allows cells to utilize glucose, which reduces blood glucose levels. The more glucose in your
blood, the more insulin the pancreas releases. Diabetes is caused by an inability to properly
process glucose, resulting in chronic hyperglycemia. Chronic hyperglycemia results in long-
term damage to various organs such as the kidneys, heart, and eyes

There are 3 primary types of diabetes. First is type 1 diabetes or can be called as insulin-
dependent diabetes mellitus or juvenile-onset diabetes. It is mainly caused by autoimmune,
genetic, and environmental factors and make or involve in the destruction of the insulin-
producing cells in the pancreas. Patients require treatment with insulin to regulate blood
glucose back to normal.

Second is type 2 diabetes or called adult-onset diabetes. Type 2 diabetes is cause by obesity,
genetic factor and age. Person who is diagnosed as type 2 diabetes will develop insulin
resistance and require increase insulin production and thus need insulin treatment. Type 2
diabetes is the most common diabetes and type 3 diabetes is gestational diabetes which
develops during pregnancy.

For diabetes patients, blood is use in order to detect or manage diabetes. Certain markers
are important in determining amount of sugar in blood or glucose level. One of those
important markers is Hemoglobin A1c (HbA1c) and by using certain tests HbA1c can be
measured and reflect the blood sugar level.

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2010

Formation of Insulin
Role of HbA1c in Diabetes Mellitus
HbA1c is a part of hemoglobin (Hb) and can best define as glucose bound to N terminal of
valine of B chain. HbA1c plays important role in Diabetes mellitus (DM) patient. HbA1c is
obtain from patient’s blood sample and tested with several laboratory tests such as
chromatography, immunoassays, and electrophoresis in order to adjust insulin dosing,
assess response to exercise and meals, and prevent hypoglycaemia

What is Hemoglobin A1c?

Hemoglobin A1c (HbA1c), also known as glycosylated hemoglobin or glycohemoglobin, is a
measure of a patient’s blood glucose level. It measure in 2-3 months. HbA1c consist of
haemoglobin A (HbA) and glucose. It forms slowly and nonenzymatically from hemoglobin
and glucose. This mechanism leads to the Amadori effect and produced a stable product
which is ketoamine that we usually know as glycosilated haemoglobin (HbA1c). It is formed
when glucose in the blood binds irreversibly to hemoglobin to form a stable complex.

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DETECTION OF DIABETES USING CHROMATOGRAPHY TECHNIQUE
2010

The hemoglobin A1 has three different subtractions which are A, B, and C. The bond
between glucose and the c-subtraction of the HB A1 is more stable. Hence, it may gives a
better information about the average glucose levels during the last 2-3 months. Each HbA1c
can lasts up to 120 days in blood based on average of red blood cells lifespan. Thus HbA1c is
only eliminated when the red cells replace. HbA1c values are directly proportional to the
concentration of glucose over approximately 2-3 months

The Hb A1c measurement can be made any time during the day even if on fasting period or
not because obviously is independent of the glucose values in that moment. Using specific
chromatography technique such as Boronate Affinity Chromatography, it can measure
HbA1c values. This test is done after a blood sample is taken from venipuncture but it can
also be performed with a capilar blood sample the same way you do at home when you
make a blood glucose test and send it to laboratory. The frequency of this lab test will vary
depending upon your type of diabetes and the individual needs of every person with
diabetes Insulin treated persons independently of their diabetes type will have their Hb A1c
measured every 3 months. Some special cases as the pregnant diabetes women will do it
every month

As we know the average span life of a red blood cell is 3 months and that is the reason why
this lab test provides us with information about the degree of diabetic control during this
period of time. An increase of 1% in the Hb A1c value shows an increase of 30 mgrs in the
average glucose levels.

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DETECTION OF DIABETES USING CHROMATOGRAPHY TECHNIQUE
2010

Ideal: < 6.5% Acceptable: 6.5 - 7.5% Poor: > 7.5%

Source from: World Health Organization (WHO)

What are factors affecting HbA1c value?

Methodological Physiological
 Hemoglobin variant and chemically  RBC kinetic – decreased RBC life span
modified Hb (carbamylated and -Decreased HbA1c
acetylated).

 Impact on reactivity N terminal  Kidney, liver disease, hemolytic

amino group B chain anemia, hemoglobinopathies and
recovery from blood loss.

 Decreased erythropoiesis – increased

HbA1c – Aplastic anemia, Iron deff.
Anemia.

 Pregnancy – HbA1c lower (decreased

RBC life span, decreased Hb)

 Inhibition of glycation decreased

HbA1c – Vit C, Vit E.

What is boronate?

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DETECTION OF DIABETES USING CHROMATOGRAPHY TECHNIQUE
2010

Boronate or boronic acid is an alkyl or aryl substituted boric acid containing a carbon-boron
bond belonging to the larger class of organoboranes. Their unique feature is that they are
capable of forming reversible covalent complexes with sugars, amino acids, and hydroxamic
acids. Thus it is an ideal option to use boronate to measure blood glucose because it can
detect and bind to sugar at HbA1c.

General structure of boronate.

What is Boronate Affinity Chromatography (BAC)?

BAC is used in Glycated haemoglobin analysis which used in the management of diabetes.
BAC is the definitive method for determination of HbA1c as it is the only interference free
method presently available to the routine laboratory. At a pH above 8, most boronate
derivatives form covalent bonds with compounds that contain cis‐diol groups in their
structure. Glycated proteins differ from non-glycated proteins by the attachment
of a sugar at various binding sites by ketoamine bond. Glycohemoglobin (GHb) thus contains
1, 2-cisdiolgroups not found in non-glycated proteins. These diol groups provide the basis
for separation of glycated and nonglycated components by boronate-affinity
chromatography.

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DETECTION OF DIABETES USING CHROMATOGRAPHY TECHNIQUE
2010

Glucose at HbA Boronate Binding of boronate

derivatives and HbA1c

Basically affinity chromatography is use to purify or separate the mixture. In this case,
affinity chromatography is used to separate glycated haemoglobin from whole blood and
can be analyze. There are three main component of affinity chromatography which is ligand,
space arm and matrix. Ligand or boronate derivatives with matrix complex are stationary
phase and sample introduced or blood is mobile phase.

1. Ligand : Site of Interaction

 molecule that binds reversibly to a specific target molecule or group of target
molecules

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DETECTION OF DIABETES USING CHROMATOGRAPHY TECHNIQUE
2010

 In this case a boronate derivative is used.

 E.g: Phenylboronic acid and Methylboronic acid]

2. Spacer: what binds ligand to support

 Carbon chain interposed between Ligand and Matrix.
 Used when active site is located deep within the spacer sample molecule
 If too long it can interact with sample species on itsown ( hydrophobic interactions)
 If too short the ligand is unable to reach the active sample molecule.

3. Matrix: Supporting Phase

 Extremely low non-specific adsorption, essential since the success of affinity
chromatography relies on specific interactions.
 Hydroxyl groups on the sugar residues are easily derivatized for covalent attachment
of a ligand, providing an ideal platform for the development of affinity media.
 An open pore structure ensures high capacity binding even for large biomolecules,
since the interior of the matrix is available for ligand attachment.
 Good flow properties for rapid separation.
 Stability under a range of experimental conditions such as high and low pH,
detergents and dissociating agents.

1. Equilibration The column
is conditioned to promote
adsorption of the target
molecule by equilibrating it
with binding buffer
Procedure of BAC
2. Sample application and
wash The sample is applied
under binding conditions.
The target molecule binds
specifically to the affinity
3. Elution the target
molecule is desorbed and
eluted by switching to
elution buffer and analyze.

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DETECTION OF DIABETES USING CHROMATOGRAPHY TECHNIQUE
2010

ligands, while all other

sample components are
washed through.

References

1. http://www.discoveriesinmedicine.com/Bar
Cod/Chromatography.html

2. http://www.chromatographyonline.com/

3. http://www.pharmaportal.com/

4. http://hplc.chem.shu.edu/NEW/HPLC_Book/index.html

5. http://www.shu.ac.uk/schools/sci/chem/tutorials

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