Diabetes and Chromatography

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2010DETECTION OF DIABETES USING CHROMATOGRAPHY TECHNIQUE

Fahmi 2007287824 1

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 re quire 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 det ect 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.

Formation of Insulin


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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 patients 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 patients 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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What are factors affecting HbA1c value?

Methodological Physiological

y Hemoglobin variant and chemicallymodified Hb (carbamylated and

acetylated).

y RBC kinetic decreased RBC life span-Decreased HbA1c

y Impact on reactivity N terminalamino group B chain

y Kidney, liver disease, hemolyticanemia, hemoglobinopathies and

recovery from blood loss.

y Decreased erythropoiesis increasedHbA1c Aplastic anemia, Iron deff.

Anemia.

y Pregnancy HbA1c lower (decreasedRBC life span, decreased Hb)

y Inhibition of glycation decreasedHbA1c Vit C, Vit E.

What is boronate?

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.


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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 Interactiony molecule that binds reversibly to a specific target molecule or group of target

molecules

y In this case a boronate derivative is used.y E.g: Phenylboronic acid and Methylboronic acid]

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


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3. Matrix: Supporting Phasey Extremely low non-specific adsorption, essential since the success of affinity

chromatography relies on specific interactions.

y Hydroxyl groups on the sugar re sidues are easily derivatized for covalent attachmentof a ligand, providing an ideal platform for the development of affinity media.

y An open pore structure ensures high capacity binding even for large biomolecules,since the interior of the matrix is avai lable for ligand attachment.

y Good flow properties for rapid separation.y Stability under a range of experimental conditions such as high and low pH,

detergents and dissociating agents.

Procedure of BAC

1. Equilibration The column

is conditioned to promote

adsorption of the target

molecule by equilibrating it

with binding buffer

2. Sample application and

wash The sample is applied

under binding conditions.

The target molecule binds

specifically to the affinity

ligands, while all other

sample components are

washed through.

3. Elution the target

molecule is desorbed and

eluted by switching to

elution buffer and analyze.


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References

1. http://www.discoveriesinmedicine.com/Bar Cod/Chromatograph

e

.html

2. http://www.chromatographe online.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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Diabetes and Chromatography