Glucose test

Introduction Glucose is monosaccharide found in fruits and also derived from the breakdown of carbohydrates in the diet & the conversion of glycogen by liver. Glucose in the bodies main source for cellular energy, brain. The glucose level in the body is maintained by insulin(cause lowering the glucose level) and glucagon, adrenalin and cortisol (cause elevation the glucose level). The various disorders in carbohydrate metabolism may be grouped in to several categories dependent primarily upon laboratory finding: those associated with a raised plasma glucose concentration (Hyperglycemia). those associated with a decreased plasma glucose concentration (Hypoglycemia).

The most frequently encountered disorder of carbohydrates metabolism is a high blood glucose due to DM. Estimates of the glucose concentration in blood are required to: Help in the diagnosis of diabetes mellitus. Management of DM patients. And monitoring of treatment in DM patients.

Methods of analysis In the past, the majority of the quantitative test 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. Enzymatic methods give “true” glucose determination because of the high specificity of an enzyme for a particular substrate and don’t consume the time, other methods contain substances dangerous for the body such as o-toludine.

Methods of analysis Reduction methods: These methods depend on the reductive property of glucose(aldose) Ferriccyanide( Hoffman’s) method. Copper sulfate method. Smogi-Nelson method. N.B. The reduction methods need alkaline medium &heat These methods are qualitative & semi-quantitative. 2. Aromatic amines methods: O-toludine +glucose (aldhyde) heat &acidity glucosamine (colored )

Trinder’s (Enz.-Dye Colorimetric ) method Methods of analysis Enzymatic methods: Hexokinase methods (The reference method). Glucose +ATP +HKADP+G6P G6P +NAD +G6PD 6 P-gluconolactone +NADH+H (measured at 340) Glucose Dehydrogenase Method:   Glucose oxidase methods Glucose +NAD GDH Gluconolactone +NADH+H (measured at 340) Kinetic method Trinder’s (Enz.-Dye Colorimetric ) method

Specimen collection 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): 2 days at 20-25 C 7 days at 2-8 C

Cont: The addition of 2mg sodium fluoride per ml blood to be collected will prevent glycolysis. 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.

There are several different types of blood glucose tests. Fasting blood sugar (FBS). This test measures blood glucose after you have not eaten for at least 8 hours. It is often the first test done to check for prediabetes and diabetes. 2-hour postprandial blood sugar. This test measures blood glucose exactly 2 hours after you start eating a meal. This is not a test used to diagnose diabetes. This test is used to see if someone with diabetes is taking the right amount of insulin with meals. Random blood sugar (RBS). It measures blood glucose regardless of when you last ate. Several random measurements may be taken throughout the day. Random testing is useful because glucose levels in healthy people do not vary widely throughout the day. Blood glucose levels that vary widely may mean a problem. This test is also called a casual blood glucose test

Principle: (Trinder’s method ) -D-glucose Mutarotase -D-glucose -D-glucose +H2O+O2 Glucose oxidase D-gluconic acid+H2O2 H2O2+ 4-aminophenazone+phenol Peroxidase Quinonemine +4 H2O The intensity of the color formed is proportional to the glucose concentration in the sample. It is determined by measuring the increase in absorbance at 500-550 nm.

Procedure: 1. Pipette into a cuvette: 2. Mix and incubate for 10 min at 37ºC. 3. Read the absorbance (A) of the samples and standard, against the Blank at 505(530-590)nm . 4. Colour is stable for 60 minutes.   Blank Standard control Sample Reagent A (mL) 1.0 Standard (µL) -- 10 Control (µL) Sample (µL) Distilled water(µL) _

4. Use beers equation to receive to the concentration of glucose in the patient sample. Glucose concentration (mg/dL)= Absorbance of Sample x Standard concentration Absorbance of Standard Conversion factor: mg/dL x 0.0555= mmol/L. Conversion factor : mmol/L * 18 = mg/dL. Conversion factor: mg/dl / 18 = mmol/L .

Normal Range: Glucose level Indication From 70 – 110 mg/dl Normal fasting glucose 110-140 mg/dl Normal 2 hrs postprandial Less than 140 mg/dl Random blood suger in diabetes mellitus, the level of fasting blood glucose is 126 mg/dl. The patient considered critically hypoglycemic if, Serum/Plasma glucose level < 45mg/dl Renal threshold for glucose is 160 – 180 mg/dl. When the blood glucose exceed the renal threshold for glucose and presences in urine reffered to glucoseuria. CSF glucose 60-90 mg/dl . Urine glucose : Nill In pregnancy, renal threshold is reduced.

ORAL GLUCOSE TOLERANCE TEST (OGTT)

Introduction On standard oral glucose dose, the response of the body regarding the absorption and metabolism of glucose said to be tolerant on meeting the normal elevation and return. Whereas abnormal and improper glucose metabolism is termed glucose intolerance. This used to diagnose diseases where the glucose metabolism is impaired as in Diabetes mellitus. Oral glucose tolerance test (OGTT) has been widely used as the golden standard for diagnosing diabetes mellitus in clinically doubtful cases. Lately, thought, the use of OGTT in primary care has been questioned for several reasons. It has low reproducibility and is very expensive. However, for the detection of diabetes in pregnant women, it is still recommended.

Patient preparation Activity--Don't smoke or exercise strenuously for 8 hours before the test or during the test. Diet--Eat a high-carbohydrate diet (> 150 g/day) for 3 days, then fast for 10 to 12 hours before the test. Don't drink coffee or alcohol for 8 hours before the test. Drugs (medicines)-Inform the person performing the test to omit any medications listed, as under taking these drugs the test results may differ (contraceptives to be stopped one cycle before the performance of OGTT). The test must be performed at daytime (morning).

Cont: Test usually takes 3 hours but can last as long as 6 hours (extended OGTT). Drink water frequently during the test (the only allowed fluid to drink). The first blood sample and the first urine sample are collected between 7 A.M. and 9 A.M., after you have fasted for 12 hours. Operator gives a test load of glucose, usually 75 – 100 gram dextrose / 300 ml water, lemon flavored . Drink the entire solution in 5 minutes.

Sample Blood samples: fasting(basal) sample, 30 minute after oral glucose load, 60min, 90min, 120min. Urine samples: first fasting urine and the hourly collected urine samples.

Calculation Glucose sample Wilkerson Criteria Fajan-conn criteria Revised Summation Fasting > 130 1 point - *If Σ of results (F + 60min. + 90 min. + 120 min.) > 600 mg/dl = Diabetic   *If Σ of results < 600 = non diabetic 30 min. 60 min. >190  ½ point >190  +1 90 min. > 165  +1 120 min. >140  ½ point > 140  +1 2 ½ hour >130  1 point Calculation of Results 2 – 3 point Diabetic ½ - 1 ½ point  Suspect Zero  Non diabetic 3  Diabetic 1 – 2 Suspect

Normal Response : A person is said to have a normal response when the 2-hour glucose level is less than or equal to 110 mg/dl. Impaired Fasting Glucose: When a person has a fasting glucose equal to or greater than 110 and less than 126 mg/dl. This is considered a risk factor for future diabetes Impaired Glucose Tolerance: A person is said to have impaired glucose tolerance when the 2-hour glucose results from the oral glucose tolerance test are greater than or equal to 140 but less than 200 mg/dl.  This is also considered a risk factor for future diabetes.

Diabetes: A person has diabetes when oral glucose tolerance tests show that the blood glucose level at 2 hours is equal to or more than 200 mg/dl. Gestational Diabetes: A woman has gestational diabetes when she is pregnant and has any two of the following: a fasting plasma glucose of more than 105 mg/dl, a 1-hour glucose level of more than 190 mg/dl. a 2-hour glucose level of more than 165 mg/dl, or a 3-hour glucose level of more than 145 mg/dl.