Colorimetric Determination of Glucose PDF

Background Dietary glucose converted to Glycogen and fatty acids stored in or and stored in the liver the adipose tissues  Cells take in glucose from blood and storing it for...

Background Dietary glucose converted to Glycogen and fatty acids stored in or and stored in the liver the adipose tissues  Cells take in glucose from blood and storing it for energy Insulin released from beta cells of pancreas  It may still be elevated, and this indicates Diabetes When glucose level >140 mg/dl or >200mg/dl after 2 hour of CHO meal or Oral Glucose Tolerance Test (OGTT). Lab diagnosis of DM based on : 1- Blood glucose level 2- Serum insulin  Specimen Collection and Preservation Individuals should Serum be fasting plasma Freshly drawn whole Freshly drawn whole blood on blood in plain tube anticoagulant (sodium fluoride) Both should be separated within 30 minutes Factors affecting stability of glucose in specimen Storage temperature, Bacterial contamination Glycolysis. glycolysis 7% in 1 hour (0.28 to 0.56 mmol/l or 5 to 10 mg/dl). Serum glucose level Unpreserved Lead to lose up 40% of their glucose urine samples after 24 hour storage at room temperature (so 5 ml of glacial acetic acid is added to the container) So, keep samples on ice during collection.  Types of glucose tests Blood glucose test Urine glucose test 1- Random blood glucose 5-Glycosylated test (RBG) hemoglobin (HbA1C) 2- Fasting blood 4- Oral glucose tolerance glucose test (FBG) test (OGTT) 3- Post prandial blood glucose (2 hour postprandial blood sugar test ) Different methods for blood glucose test: 1- Random blood glucose test Measures the blood glucose level at any time and required in an emergency. 70 - 140 mg/dl in an average adult 140 - 200 mg/dl is considered pre-diabetes.  200 mg/dl is considered diabetes  Different methods for blood glucose test: 2-Fasting blood glucose test Measuring after overnight fast (10 hours at least). FBG is better than RBG Clinical significance: Less than 100 mg/dl is Normal 100- 125 mg/dl is considered pre-diabetes.  >125mg/dl is considered diabetes  Different methods for blood glucose test: 3- Postprandial blood glucose test (Two hour postprandial blood glucose test) Measuring exactly 2 hours after eating a meal. Indication: For monitoring the blood glucose in diabetic patients It can see if you are taking right dose of insulin with meal NORMAL Level : < 140 mg/dl Different methods for blood glucose test: 4- Oral glucose tolerance test (OGTT)  Is a medical test measures the body's ability to use a type of sugar.  Standard dose of glucose is ingested by mouth and blood levels are checked two hours later.  Different methods for blood glucose test: 4- Oral glucose tolerance test (OGTT) Indication: 1- Borderline fasting or postprandial blood glucose. 2- persistent glucosuria 3- glucosuria in pregnant women 4- Pregnant women with a family history of diabetes mellitus.  Blood samples is first taken after overnight fast  The patient is asked to drink 75 g glucose in 300 ml water (children 1.75 g/kg body weight) with in 5 min  Then plasma glucose level are measured every 30 min wait for 2 hours  Urine samples can be tested for glucose at time 0 and after 2 hours  Normal glucose levels in OGTT  Fasting plasma glucose below 95 mg/dl.  The 1 hour OGTT glucose level below 180 mg/dl  The 2 hour OGTT glucose level below 155 mg/dl  Clinical significance of OGTT  Normal less than 140 mg/dl.  Pre-diabetes 140-199 mg/dl  Glucose levels above 200 mg/dl at 2 hours confirms Different methods for blood glucose test: 5- Glycosylated hemoglobin (Hemoglobin A1c or HbA1c) Measures the amount of glucose attached to hemoglobin this means the hemoglobin has become glycosylated.  An HbA1c test shows what the average amount of glucose attached to hemoglobin has been over the past three months. So  People who have diabetes need this test regularly to see if their levels are staying within range.  And It can tell if you need to adjust diabetes medicines. Clinical significance of A1c Test Clinical significance of A1c Test  Normal less than 4%- 5.6%  Pre-diabetes 5.7%-6.4%  Diabetes more than 6.5%  Good diabetic control less than 7%  Fair diabetic control 7-8%  Poor diabetic control more than 8%  Reference ranges of blood glucose levels Normal Pre-diabetes Diabetes (mg/dl) (mg/dl) (mg/dl) Fasting < 100 100-125 >126 RBG < 140 140-199 >200 A1C test 6.5% OGTT 200  Urine glucose test. The presence of glucose in the urine is called Glucosuria. Occurs when BG conc. > 180 mg/dl.  Colorimetric determination of glucose by enzymatic method The more recent technique, less susceptible to this kind of error Using enzymes specific to glucose The two most common employed enzymes are Glucose oxidase Hexokinase.  Method GOD-PAP enzymatic colorimetric method.  GOD (glucose oxidase )  PAP ( peroxidase)  Assay Principle A red violet dye as indicator  Procedure Reagents Glucose standard (St) 100 mg/dL 5.55 mmol/L Reagent (R): Phosphate Buffer 100 mmol/L Phenol 4.0 mmol/L 4-amino-antipyrine 1.0 mmol/L Glucose oxidase > 20 KU /L Peroxidase > 2.0 KU/L Sodium Azide 8 mmol/L Note: Reagent (R) contains sodium azide which may react with copper or lead plumbing.  Procedure  System Parameter  Wavelength: 546 nm (492 – 550 nm)  Optical path: 1 cm  Assay type : Endpoint  Direction : Increase  Sample : Reagent Ratio 1: 100  Incubation time : 20 minutes at 20 – 25 oC or 10 minutes at 37 oC  Zero adjustment :Reagent blank  Sensitivity: 5 mg/dL (0.27 mmol/L)  Linearity: 500 mg/dL ( 27.7 mmol/L)  Procedure  Method Blank Standard Sample Reagent (R) 1.0 ml 1.0 ml 1.0 ml Standard -------- 10 μl ------ Sample ----------- ------- 10 μl  Mix and incubate for 10 minutes at 37 oC or 20 minutes at 15 -25oC.  Measure absorbance of specimen (A specimen) and standard (A standard) against reagent blank within 30 minutes.  Procedure  Calculation Please note: Large amounts of reducing substances as ascorbic acid, creatinine, glutathione and uric acid react with hydrogen peroxide and stimulate low glucose concentration  Procedure  Clinical significance Expected Values Human, dog plasma (fasting) 70 – 120 mg/100 ml. Ruminants 40-60 mg/100 ml. Urine Random 5.0 - 15 mg/100 mg.  If blood glucose >110 mg/dl-- Hyperglycemia Causes: Diabetes mellitus Acute stress Adrenal hyperactivity (Cushing's syndrome) Hyperthyroidism Pancreatic cancer or pancreatitis Drugs: e.g. corticosteroids If blood glucose