BLOOD GLUCOSEGlucose comes from carbohydrate foods. It is the main source of energy used by the body.

Brain cells are very dependant on glucose for their energy supply: hypoglycemia is likely to impair cerebral function. This is because they cannot:

Store glucose in significant amounts, Synthesize glucose, Metabolize substrates other than glucose and ketones. Extract enough glucose from extracellular fluid at low concentrations for their metabolic needs, because

entry into brain cells is not facilitated by insulin.

Normally the plasma glucose concentration remains between about 80 and 200 mg/dl (4.5 and 11 mmol/L) despite the intermittent load entering the body from the GIT. The maintenance of plasma glucose concentrations below about 200 mg/dl minimizes loss from the body as well as providing the optimal supply to the brain. Renal tubular cells reabsorb almost all the glucose filtered by the glomeruli and urinary glucose concentration is normally too low to be detected by the usual tests, even after a carbohydrate meal. Significant glycosuria only occurs if the plasma glucose concentration exceeds about 200 mg/dl – the ‘renal threshold’.

Types of blood glucose tests:

• Fasting blood sugar (FBS) measures blood glucose after fasting for at least 8 hours. It often is the first test done to check for diabetes.

Normal Range: 80 – 126 mg/dl (4.5 – 7.0 mmol/L)

• 2-hour postprandial blood sugar measures blood glucose exactly 2 hours after eating a meal. Its also called Random Blood Sugar (RBS.)

Normal Range: 126 – 200 mg/dl (7.0 – 11.1 mmol/L)

• Oral glucose tolerance test is used to diagnose prediabetes and diabetes. An oral glucose tolerance test is a series of blood glucose measurements taken after drinking a sweet liquid that contains glucose. This test is commonly used to diagnose diabetes that occurs during pregnancy (gestational diabetes).

Glucose tolerance test determines the ability of an individual to utilize a given quantity of glucose.

In normal individual following a meal, there is a temporary rise in the blood glucose level with a return to fasting level within 2-3 hours. Glucose tolerance test determines the degree and duration of hyperglycemia after an oral or intravenous administration of a known quantity of glucose.

PROCEDURE:

The test is best carried out in the morning after an overnight fasting.

Blood is taken for the determination of fasting blood glucose and a specimen of urine is collected.

Glucose is administered orally in a dose of about 1.75g/kg bodyweight up to a maximum of 75g dissolved in about 250-300ml of water.

This is best followed by about 50-100ml of water which takes away the sweet taste and lessens the risk of vomiting.

Blood for the estimation of blood glucose is taken at half hourly intervals, for two and half hours after the glucose solution has been drunk

Urine specimens are collected at intervals.

DETERMINATION OF BLOOD GUCOSE LEVEL

Blood glucose can be estimated by any of the following methods;

Folin and Wu’s method

Nelson and Somogyi’s method

O-toluidine method

O-toluidine method is simple and specific and most widely used method for the determination of blood glucose.

DETERMINATION OF GLUCOSE LEVEL IN URINE

Glucose can be detected in the urine by Benedict’s test.

Why It Is Done

Blood glucose tests are done to:

• Check for diabetes.

• Monitor treatment of diabetes.

• Check for diabetes that occurs during pregnancy (gestational diabetes).

• Determine if an abnormally low blood sugar level (hypoglycemia) is present.

TYPES OF RESPONSES TO OGTT

Following are the responses seen in OGTT;

1. Normal Response Initial zero hour fasting blood glucose is within the normal range and the maximum

blood glucose value reaches either half-hour or 1 hour after taking the glucose. The blood glucose then returns rapidly to the normal fasting limit which often

reaches in 1 and a half hours and almost always at 2 hours.By then, it should be below 120mg%

Urine samples collected during test should be free of glucose.2. Abnormal responses;

a. Impaired glucose tolerance;

Glucose tolerance test diminishes to a greater extent in diabetes mellitus. The most significant finding in the diagnosis of diabetes is the failure of blood glucose level to

fall below 120mg even by 2 hours. The peak level is frequently above normal curve and fasting level may or may not be raised. In a diabetic there may or may not be glycosuria in the fasting urine but the post glucose urine

definitely contains sugar. The GTT response in mild and severe diabetes is shown in the following figures;

b. Renal Glycosuria;

The curve is normal but one or more samples of urine contain glucose. When the blood levels are elevated, the glomerular filtrate contains more glucose than can be

absorbed, so the excess passes into the urine to produce glycosuria. Normally glycosuria occurs when venous blood concentration exceeds 180-200mg%. This is

termed as renal threshold for glucose. This type of curve is harmless and patients are not likely to develop diabetes.

c. Lag type of tolerance curve;

Some otherwise normal individuals show an exaggerated rise in blood glucose, following an oral load but the level quickly falls and the two hours concentration is within normal limits.

Transient glycosuria usually occurs. This phenomenon usually results from an increased rate of glucose absorption from the gut,

following rapid emptying of stomach and occurs in some hyperthyroid patients. The increase in blood glucose level is due to delay in insulin mechanism coming into action

d. The flat curve of increased glucose tolerance;

In normal subjects given a glucose load, blood glucose levels rise to a peak at 30or at 60 min and then fall to near-fasting levels at two hours but some individuals show ver5y little rise.

Flat curves are seen in patients with hypo activity of other endocrine organs, e.g., in hypopituitarism and Addison’s disease, in malabsorption and in many normal subjects

GLYCATED HEMOGLOBIN

Glycated hemoglobin provides an accurate and objective measure of glycemic control over a period of weeks to months.

This can be utilized as an assessment of glycemic control in a patient with known diabetes, but is not sufficiently sensitive to make a diagnosis of diabetes and is usually within the normal range in patients with impaired glucose tolerance

HYPERGLYCEMIA

Elevated blood glucose level is known as Hyperglycemia. Fasting hyperglycemia is highly suggestive of diabetes

Causes

Conditions other than diabetes in which hyperglycemia is present include;

Hyperthyroidism Hyperpituitarism Hyperactivity of adrenal cortex and medulla Emotional stress

These changes cause small increases not exceeding 150mg/100ml

Other causes include infectious diseases and intra cranial diseases such as meningitis, encephalitis, tumours and hemorrhage.

HYPOGLYCEMIA

It is a condition in which blood glucose is below 40mg/100ml.

Causes

Over dosage with insulin or oral hypoglycemic drugs in diabetic patients. Insulin secreting tumors of pancreas Hypothyroidism Hypopituitarism

Addison’s disease Liver disease, such as hepatitis. Malnutrition Severe exercise Sepsis

Diabetes Mellitus

Diabetes is a disease in which the body doesn't produce or properly use insulin.

Types

Type 1 diabetes (previously known as insulin-dependent or childhood-onset) is characterized by a lack of insulin production. Without daily administration of insulin, Type 1 diabetes is rapidly fatal.

Type 2 diabetes (formerly called non-insulin-dependent or adult-onset) results from the body’s ineffective use of insulin. Type 2 diabetes comprises 90% of people with diabetes around the world, and is largely the result of excess body weight and physical inactivity.

Gestational diabetes is hyperglycaemia which is first recognized during pregnancy.

“Pre-diabetes” is a condition in which blood glucose levels are higher than normal but not yet diabetic. People with pre-diabetes are at increased risk for developing type 2 diabetes, heart disease and stroke. This condition are as follows:

Impaired Glucose Tolerance (IGT) and Impaired Fasting Glycaemia (IFG) are intermediate conditions in the transition between normality and diabetes. People with IGT or IFG are at high risk of progressing to type 2 diabetes, although this is not inevitable.

Signs and Symptoms

Cardinal Signs : 3 P’s

1. Polyuria – frequent urination

2. Polydipsia – displays of excessive thirst

3. Polyphagia – excessive hunger or eating

Weight loss

Blurred Vision

Slow wound healing

Infections: pyorrhea (periodontal infections), urinary tract infection/, vasculitis, cellulites, furuncle, carbuncles, vaginal infections

Weakness and paresthesia

Signs of inadequate circulation of the feet

Signs of accelerated atherosclerosis (renal, cerebral, cardiac, peripheral)

Type 1- Symptoms include excessive excretion of urine (polyuria), thirst (polydipsia), constant hunger, weight loss, vision changes and fatigue. These symptoms may occur suddenly.

Type 2 symptoms

Symptoms may be similar to those of Type 1 diabetes, but are often less marked. As a result, the disease may be diagnosed several years after onset, once complications have already arisen.

Until recently, this type of diabetes was seen only in adults but it is now also occurring in obese children.

Gestational Diabetes

Symptoms of gestational diabetes are similar to Type 2 diabetes. Gestational diabetes is most often diagnosed through prenatal screening, rather than reported symptoms.

ESTIMATION OF SERUM GLUCOSE

(By Glucose Oxidase – Peroxidase Method)

PRINCIPLE:

The reaction system is as follows:

Glucose + O2 + H2O GOD Gluconic acid + H2O2

2H2O2 + 4-AP + 4-hydroxybenzoate POD red quinoneimine

PROVIDED REAGENTS:

Standard: 1 g / L or 100 mg/ dl glucose solution.

WHO Criteria for diagnosing Diabetes:

Reagents: Solution containing glucose oxidase (GOD), per-oxidase (POD), 4- Aminophenazone (4-AP) and phosphate buffer pH 7.0 containing hydroxybenzoate, in the following concentrations:

GOD_____________________ ≥ 10KU/l

POD_____________________ ≥ 1KU/l

4-AP_____________________ 0.5Mm

Phosphate_________________ 100mM, pH 7.0

Hydroxybenzoate___________12mM

STABILITY AND STORAGE INSTRUCTIONS:

Provided Reagents: stable in refrigerator (2-10 0C). Do not expose to high temperature for longer periods of time.

INSTABILITY OR DETERIORATION OF REAGENTS:

During its use, the reagent may develop a light pink coloration which does not affect its performance as long as Blank is processed for each determination lot and a standard periodically. Discard when the Blank readings are higher than 0.160 O.D. or the standard readings are abnormally low.

SAMPLE: Serum, plasma, whole blood or CSF.

a. Collection: Obtain serum in the usual way or plasma collected with ordinary anti-coagulant. Tests can also be performed on other biological fluids such as CSF. The test can be performed on capillary blood when venous blood cannot be drawn, or in case of an emergency.

b. Additives: If plasma is used as sample, it is recommended to use Weiner labs’ Anticoagulant G or heparin.c. Known Interfering substances: Sera or plasma with visible or intense hemolysis should be deprotienized. No interferences are observed from bilirubin up to 100mg/ dl, triglycerides up to 5g/ L, and hemoglobin up to 0.35 g/ dl.

STABILITY AND STORAGE INDICATIONS:

Enzymatic destruction of blood glucose by RBC’s and leucocytes is proportional to the temperature at which blood is stored, reaching its maximum at 37 0C. This process is not inhibited by freezing, thus blood should be centrifuged within 2 hours after collection. The clear supernatant must be transferred to another tube for storage. In this manner, glucose is stable for 4 hours at room temperature or 24 hours refrigerated. When it is not possible to process the sample as indicated above, add a preservative to the blood when collecting.

REQUIRED MATERIAL:

Spectrophotometer. Micropipettes and pipettes for measuring the stated volumes. Spectrophotometer cuvettes or Photo colorimeter tubes. Water bath at 37 0C. Watch or timer.

ASSAY CONDITIONS:

Wave-length: 505nm in spectrophotometer, 490-540-nm for photo colorimeter with green filter.

Reaction temperature: 37 0C.

Reaction time: 5 min.

Sample volume: 10 µl (0.01 ml).

Reagent volume: 1000 µl (1ml).

Final reaction volume: 1.01 ml.

PROCEDURE:

B S U

Standard - 10 µl -

Sample - - 10 µl

Reagent 1.0 ml 1.0 ml 1.0 ml

STABILITY OF FINAL REACTION:

Final reaction color is stable for 30 min, thus absorbance should be read within that period.

OBSERVATIONS & CALCULATIONS

O.D. of sample: ____________

O.D. of standard: ___________

Concentration of standard: 100 mg/ dl

Serum glucose concentration = O.D. of sample

× Concentration of standard

O.D. of standard

Serum glucose concentration is: ____________ mg/dl