Glucose Determination Mohammed Laqqan

Introduction 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 Mohammed Laqqan

a decreased plasma glucose concentration (hypoglycemia). 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. Mohammed Laqqan

Measuring blood glucose is used for the diagnosis of: Carbohydrate metabolism disorders Monitoring of treatment in diabetes mellitus Neonatal hypoglycemia, Idiopathic hypoglycemia, Insulinoma. Hypoglycemia: is the most common metabolic problem in neonates. In children, a blood glucose value of less than 40 mg/dL (2.2 mmol/L) represents hypoglycemia. A plasma glucose level of less than 30 mg/dL (1.65 mmol/L) in the first 24 hours of life and less than 45 mg/dL (2.5 mmol/L) thereafter constitutes hypoglycemia in the newborn. Patients with hypoglycemia may be asymptomatic or may present with severe central nervous system (CNS) and cardiopulmonary disturbances. The most common clinical manifestations can include altered level of consciousness, seizure, vomiting, unresponsiveness, and lethargy. The causes in neonates include the following (see Etiology): Inappropriate changes in hormone secretion Inadequate substrate reserve in the form of hepatic glycogen Inadequate muscle stores as a source of amino acids for gluconeogenesis Inadequate lipid stores for the release of fatty acids An insulinoma is a tumor of the pancreas that is derived from beta cells and secretes insulin. Insulinoma : Beta cells secrete insulin in response to increases in blood glucose. The resulting increase in insulin acts to lower blood glucose back to normal levels at which point further secretion of insulin is stopped. In contrast, the secretion of insulin by insulinomas is not properly regulated by glucose and the tumors will continue to secrete insulin causing glucose levels to fall further than normal. Signs and Symptoms Patients with insulinomas usually develop neuroglycopenic symptoms. These include recurrent headache, lethargy, diplopia, and blurred vision, particularly with exercise or fasting. Severe hypoglycemia may result in seizures, coma, and permanent neurological damage. Mohammed Laqqan Mohammed Laqqan

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. Mohammed Laqqan

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. Mohammed Laqqan

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 nm Mohammed 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

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. Mohammed Laqqan Mohammed Laqqan

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 Mohammed Laqqan Pre-Diabetes:is a condition in which blood glucose levels are higher than normal but are not high enough for a diagnosis of diabetes. People with pre-diabetes are at an increased risk for developing type 2 diabetes and for heart disease and stroke. Symptoms of Diabetes Mellitus: Frequent urination Excessive thirst Dry Skin Slow healing of cuts Blurry eyesight Always feeling hungry Feeling tired and weak Weight loss Skin infections Why these symptoms occur:  When blood glucose rises above a certain level, it is removed from the body in urine. A resulting increase in the volume of urine occurs due to the osmotic effect of glucose. Picture the kidney as a dam: when there is too much glucose in the blood, the excess is diluted with water and spills out. The maximum blood glucose level reached before sugar spills out is called the kidney threshold and is usually about 180 mg/ml. This results in two conditions: polyuria or excessive urination and glycosuria or glucose in the urine. The loss of water through urination triggers the brain to send a message of thirst. This results in a condition known as polydipsia or excessive thirst. Excessive urination can also lead to dehydration, which accounts for the dry, itchy skin. The blurred vision can be caused by fluctuations in the amount of glucose and water in the lenses of the eyes. As far as always feeling hungry this is because there is no insulin or the insulin is unable to activate the uptake of glucose into the cells and therefore the cells are starving. This triggers the brain to send a message of hunger, resulting in polyphagia or excessive hunger. The fact that the cells are low on energy also explains why there are feelings of fatigue and weakness. Weight loss may occur in people due to the inability to supply the cells with the needed amount of glucose. The complication of skin infections occurs at times because of the excess glucose in the blood, this excess glucose suppresses natural defense mechanisms such as the activity of white blood cells and the glucose is also an excellent food source for bacteria. 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. Mohammed Laqqan

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

high-performance liquid chromatography immunoassay 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 Mohammed Laqqan Mohammed Laqqan

Normal results 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