February 11, 2010

DETECTION OF DIABETES USING CHROMATOGRAPHY TECHNIQUE

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 diabet es or can be called as insulindependent 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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February 11, 2010

DETECTION OF DIABETES USING CHROMATOGRAPHY TECHNIQUE

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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February 11, 2010

DETECTION OF DIABETES USING CHROMATOGRAPHY TECHNIQUE

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 thi s 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.

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

Source from: World Health Organization (WHO)

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February 11, 2010

DETECTION OF DIABETES USING CHROMATOGRAPHY TECHNIQUE

What are factors affecting HbA1c value?

Methodological y Hemoglobin variant and chemically modified Hb (carbamylated and acetylated). y

Physiological RBC kinetic decreased RBC life span

-Decreased HbA1c

y

Impact on reactivity N terminal amino group B chain

y

Kidney, liver disease, hemolytic anemia, hemoglobinopathies and recovery from blood loss.

y

Decreased erythropoiesis

increased

HbA1c Aplastic anemia, Iron deff. Anemia.

y

Pregnancy

HbA1c lower (decreased

RBC life span, decreased Hb)

y

Inhibition of glycation decreased HbA1c 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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February 11, 2010

DETECTION OF DIABETES USING CHROMATOGRAPHY TECHNIQUE

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 y molecule that binds reversibly to a specific target molecule or group of target molecules y y In this case a boronate derivative is used. E.g: Phenylboronic acid and Methylboronic acid]

2. Spacer: what binds ligand to support y y y y 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.

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February 11, 2010

DETECTION OF DIABETES USING CHROMATOGRAPHY TECHNIQUE

3. Matrix: Supporting Phase y 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 attachment of 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 y Good flow properties for rapid separation. 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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February 11, 2010

DETECTION OF DIABETES USING CHROMATOGRAPHY TECHNIQUE

References 1. http://www.discoveriesinmedicine.com/Bar Cod/Chromatograph .html 2. http://www.chromatograph 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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