• All body tissues can utilize glucose, the principle and almost exclusive carbohydrate circulating in blood.

• Glucose is a reducing monosaccharide that serves as the principal fuel of all the tissues. It enters the cell through the influence of insulin and undergoes a series of chemical reactions to produce energy.

• 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

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Introduction

• 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);

– a decreased plasma glucose concentration (hypoglycemia).

• Estimates of the glucose concentration in blood are required to help in the diagnosis of diabetes mellitus or any other condition in which there is abnormal carbohydrate metabolism in the body.

• Lack of insulin or resistance to its action at the cellular level causes diabetes .

Mohammed Laqqan

Mohammed Laqqan

Purposes of Determination

• Methods for the quantitative measurement of glucose in blood were introduced at the beginning of the century.

• The principle use of the test is for: – the diagnosis and management of diabetes, – but it is absolutely essential for the detection and

proper management of hypoglycemia, a condition encountered much less frequently.

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• Measuring blood glucose is used for the diagnosis of:

– Carbohydrate metabolism disorders – Monitoring of treatment in diabetes mellitus– Neonatal hypoglycemia, – Idiopathic hypoglycemia, – Insulinoma.

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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.

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• 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.

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Methods of glucose analysis• 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: Alkaline ferricyanide (quantitative)

– involves the reduction of yellow ferricyanide, Fe(CN)6, to colorless ferrocyanide, Fe(CN)6, by glucose in alkaline conditions.

• Method 3: 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 nmMohammed Laqqan

Enzymatic Methods

• Method 4: 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. (340nm)

• Method 5: Glucose oxidase coupled reaction, (quantitative)

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

Mohammed Laqqan

Mohammed Laqqan

Glucose Oxidase Method • Glucose present in the plasma is oxidized by the

enzyme glucose oxidase (GOD) to gluconic acid with the liberation of hydrogen peroxide, which is converted to water and oxygen by the enzyme peroxidase (POD).

• 4 aminophenazone, an oxygen acceptor, takes up the oxygen and together with phenol forms a pink coloured chromogen.

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Specimen collection and preparation • Serum, plasma is suitable for samples. • 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

• Note: Whole blood and hemolysis are not recommended for use as a sample.

• Fluoride and iodoacetate have been used as inhibitors of glycolysis to preserve blood that cannot be separated rapidly.

Mohammed Laqqan

Mohammed Laqqan

Glycated Hemoglobin (HbA1C)

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

• It is estimated 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.

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• Glycated hemoglobin is a form of hemoglobin which is measured primarily to identify the average plasma glucose concentration over prolonged periods of time.

• It is formed in a non-enzymatic glycation pathway by hemoglobin's exposure to plasma glucose.

• Normal levels of glucose produce a normal amount of glycated hemoglobin.

• As the average amount of plasma glucose increases, the fraction of glycated hemoglobin increases in a predictable way.

Mohammed Laqqan

Mohammed Laqqan

• 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 two months.

• Today, hemoglobin A1C testing is performed to monitor diabetics suspected of having irregular control over their glucose level.

• There are a number of techniques used to measure A1C. Laboratories use:

high-performance liquid chromatography immunoassay

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• An HbA1c of 6% or less is normal.• If your HbA1c is above 6.5% you may be diagnosed

with diabetes.• If you have diabetes, try to keep your HbA1c level at

or below 7%. However, you and your health care provider must decide what a normal HbA1c level is for you.

Mohammed Laqqan

Normal results