Glucose and Glycated Hb( HbA1C )



Glucose and Glycated Hb( HbA1C )

Introduction:

- 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.

Disorders in Carbohydrate Metabolism:

- The various disorders in carbohydrate metabolism may be grouped into several categories dependent primarily upon laboratory findings:

o those associated with a raised plasma glucose concentration (hyperglycemia).

o Those associated with decreased plasma glucose concentration (hypoglycemia).

Estimates of the Glucose Concentration:

- Estimates of the glucose concentration in blood are required to:

1. help in the diagnosis of diabetes mellitus.

2. management of DM patients.

3. and monitoring of treatment in DM patients.

4. It is also essential for the detection and proper management of hypoglycemia, a condition encountered much less frequently.

5. neonatal hypoglycemia.

6. or any other condition in which there is abnormal carbohydrate metabolism in the body.

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.

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.

Method 3: Glucose oxidase coupled reaction:

- Quantitative.

- One of the most frequently used specific glucose methods uses two coupled enzyme reactions.

Specimen Collection:

- 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.

| [pic] |sodium fluoride Potassium oxalate/ |For glucose determinations Oxalate and EDTA |

| |• Sodium fluoride/ Na2 EDTA |anticoagulants will give plasma samples. Sodium |

| |• Sodium fluoride (serum tube) |fluoride is the |

| | |antiglycolytic agent. |

| | |Tube inversions ensure proper mixing of additive |

| | |with blood. |

Specimen Collection And Preparation:

- 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) :

o 2 days at 20–25oC

o 7 days at 2–8oC

Glycated Hb ( HbA1C)

Introduction:

- Diabetes mellitus, as previously stated is a condition of hyperglycemia.

- It is estimated that 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 life-span of the person afflicted.

- 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.

Glycosylated Hemoglobin:

- In adults, hemoglobin is a mixture of three forms:

o Hb A1, Hb A2, and Hb F, with Hb A1 predominating.

- Hemoglobin A1 consists of three subforms:

o 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

- 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;

o the mean daily blood glucose concentration.

o and the degree of carbohydrate imbalance over the preceding period (90-120 days) .

- 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.

Significance of Test:

- 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,

- The glycosylated hemoglobin is irreversible; it shows what type of diabetic control has occurred over several months.

Method of Analysis:

- HPLC: ion- exchange chromatography

- Principle of Analysis:

o Spectral absorbance of separated Hb components. Measure HbA1C routinely included an agent in the hemolysis reagent to remove the labile GHb.

Specimen:

- 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.

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