glucose estimation in a biochemistry lab.pptx

glucose estimation in a biochemistry lab.pptx

• 1.
  GLUCOSE ESTIMATION
• 2.
  SPECIMEN COLLECTION ANDSTORAGE • Glycolysis decreases serum glucose by 5-7% in one hour(5-10 mg/dl) in normal uncentrifuged blood at room temperature • The rate of in Vitro glycolysis is higher in the presence of leukocytosis or bacterial contamination • Cell free sterile plasma has no glycolytic activity • In seperated, non hemolyzed sterile serum; glucose concentration is stable for 8 hours at 25 Celsius and upto 72 hours at 4 Celsius
• 3.
  • Naf isused to inhibit glycolysis by inhibiting Enolase, an enzyme that requires mg2+ • Fluoride ions in high concentration inhibit the activity of urease, so those specimens can’t be used for direct assay • Potassium oxalate causes loss of water and dilutes the sample • Acidification of blood using citrate buffer inhibit in vitro glycolysis more effectively than flouride
• 4.
  • Inhibitors ofglycolysis are necessary in patients with generally increased leukocytes counts because differences of upto 65mg/dl are observed • In 24 hours collection of urine, 5 ml of glacial acetic acid is added as preservative • Urine should be stored at 4 Celsius • Urine samples may lose as much as 40% of initial glucose after 24 hours at room temperature
• 5.
  Glucose estimation methods •Hexokinase method : reference method • Glucose = glucose 6 phosphate = 6 phosphogluconate • The amount of reduced nadp or Nadh produced is directly proportional to glucose in sample • This is measured by increase in absorbance at 340 nm • In the reference method, serum or plasma is deproteinised by addition of barium hydroxide and zinc sulphate
• 6.
  • Alternative approach:naf with anticoagulant like edta, heparin ,oxalate or citrate is used • Hemolysed specimens of more than 0.5 mg/dl are unsatisfactory • Other source of interference: drugs,billurubin, lipemia(>500mg/dl) • The linearity of this method is 0-500mg/dl
• 7.
  • Glucose oxidasemethod: • Oxidation of glucose to glauconic acid and hydrogen peroxide ; chromogenic o2 acceptor such as O-DIANISIDINE results in formation of coloured compound • Glucose oxidase is highly specific for beta-D-glucose • Alpha (36%) and beta(64%) are in solution and so to change to beta form ; MUTAROTASE enzyme is added
• 8.
  • Substances suchas uric acid, bilirubin, hb, tetracycline, glutathione inhibit the reaction leading to lower values • Catalase activity decomposes peroxide and decrease the final Color • This method produces falsely low results in urine samples because of uric acid • Urine should be treated first with ion exchange resin to remove interfering substances
• 9.
  • In dry,multilayer, slide automated system, glucose oxidase method is used • Reflectance spectrophotometry
• 10.
  • Glucose dehydrogenasemethod: • This enzyme catalyses the oxidation of glucose to gluconolactone with concomitant reduction of nad+ to nadh • This reaction is highly specific for glucose
• 11.
  Measurement of glucosein urine • Examination of urine for glucose provides no information if the glucose concentration is below renal threshold (180mg/dl) • Benedicts method is qualitative method for total reducing sugars • Clinistix, diastix, chemstrip use glucose specific enzyme oxidase in chromogen assay; which are semi quantitative assays • Clinistix – 100mg/dl or more • Automatic urine analysis systems – 250mg/dl • Ketone bodies,ascorbic acid and salicylates gives false negative results • Keto-diastix and diascreen tests are for both glucose and ketone bodies