Methods of Glucose measurement Flashcards
Specimen considerations for glucose measurement to prevent glycolysis
Use of NaF or SST
Rate of glycolysis at room temperature if not prevented
7 mg/dL/h
Rate of glycolysis at 4°C if not prevented
2 mg/dL/h
Effect of 10% contamination with 5% dextrose
Artificially elevates glucose levels
Initial reaction of chemical glucose methods
Alkaline copper titrate (Benedict’s method)
Reaction involved in Folin-Wu method
Cu+ + phosphomolybdic acid → phosphomolybdenum (blue color)
Reaction involved in Nelson-Somogyi method
Cu+ + arsenomolybdic acid → arsenomolybdenum
Reaction involved in Neocuproine method
Cu+ + neocuproine → Cu-neocuproine complex (yellow/yellow-orange color)
Principle of Ferric Reduction (Hagedorn-Jensen method)
Ferricyanide (yellow-orange) reduced to ferrocyanide (colorless) by glucose
Reaction in condensation method (Dubowski method)
Glucose + o-toluidine → Schiff’s base (green color)
Glucose oxidase reaction in enzymatic method
β-D-glucose + O2 + H2O → Gluconic acid + H2O2
Detection method for glucose oxidase
Peroxidase-coupled (Trinder reaction)
End product of glucose oxidase detection (Trinder reaction)
Oxidized chromogen (quinonimine; red/red-purple) + H2O
Interference causing false increased glucose in Trinder reaction
Oxidizers
Interference causing false decreased glucose in Trinder reaction
Reducing agents (drug metabolites, ascorbic acid, ketoacids)
Polarographic method measures
Degree of O2 consumption using a pO2 Clark electrode
Substances required in polarographic methods (MICE)
Molybdate, Iodate, Catalase, Ethanol
Hexokinase reaction for glucose measurement
Glucose + ATP → Glucose-6-PO4 + ADP
Coupling reaction in hexokinase method
Glucose-6-PO4 + NADP → 6-phosphogluconolactone + NADPH
Detection of hexokinase reaction
Increased absorbance at 340 nm (NADPH)
Interference in hexokinase method
Hemolyzed/icteric samples cause false decreased glucose estimate
Glucose dehydrogenase reaction
Glucose + NAD → Gluconolactone + NADH + H+
Alternative detection for glucose dehydrogenase method
NADH + tetrazolium dye → formazan blue (colorimetric-spectrophotometric measurement)
Specificity of glucose dehydrogenase method
Highly specific for glucose, not subject to interference from substances in serum
Application of glucose dehydrogenase method
Primarily used in POCT devices (glucometers)
Reference method for glucose measurement
Hexokinase (UV method)
UV absorbance wavelength for glucose dehydrogenase/hexokinase
340 nm
Advantage of glucose dehydrogenase
Not subject to common interferences, highly specific
