Monday, November 19, 2012

BLOOD GLUCOSE MEASUREMENT


Measured in plasma or whole blood. Plasma values are slightly lower than whole blood. Glucose measurement in urine. In urine the blood glucose ranges in around 180 mg/dl. Serum or plasma is the specimen of choice. For urine 24 hr collections can be done and should be preserved by adding 5 mL of glacial acetic acid in vial. Other preservatives lik 5g sodium benzoate can be used.

Hexokinase and glucose oxidase are the two main types of method used to measure glucose in body fluids.

HEXOKINASE METHOD:

(Source: Tietz clinical chemistry, 4th Edition)

The rate of increase in absorbance by NADH is measured at 340 nm. This is the reference method. Specimen blank is used to correct for interferences. The interfering substances are bilirubin, drugs, lipemia, hemolysis. 

GLUCOSE OXIDASE METHOD:

(Source: Tietz clinical chemistry, 4th Edition) 
  
Glucose oxidase is highly specific for beta-D-glucose. About 36% and 64% of glucose in solution are in alpha and beta form respectively. Complete reaction of glucose requires mutarotation of alpha to beta. Reagents contain mutarotase for this. Otherwise extended incubation time allows spontaneous conversion.
In second step various substances, like uric acid, ascorbic acid, bilirubin, hemoglobin, tetracycline, and glutathione inhibit the reaction by competing with the chromogen for hydrogen peroxide producing lower values. Incroporation of potassium ferrocyanide decreases interference by bilirubin. Incorporation of substituted phenol compounds like dimethyl alanine, danisidine, increases the absorptivity and redox reaction. This type of coupled reaction by using substituted benzene compounds to enhance absorptivity and redox system is known as the trinder reaction
Older procedure use quantative Benedict’s test to determine the glucose in blood and are based on its property of acting as reducing agent in hot alkaline solution. In hot alkaline condition the aldehyde group of glucose readily reduces cupric ions to cuprous ions, which form cuprous oxide. Sodium sulphate is used in the reagent to decrease the solubility of oxygen in reagent otherwise this oxygen will again reoxide the cuprous oxide.
In folin wus method proteins are precipitated with tungstic acid and the clear protein free filtrate is used in the reaction with cupric ions.
In somogyi-Nelson method proteins are precipitated by addition of Ba(OH)2 and ZnSO4. Protein is removed as zinc proteinate sulfhydryl compounds as zinc salts and the remaining zn and Ba ions as zinc hydroxide and barium sulfate. Before the advent of enzyme assay, this was the reference method for glucose.
Another test is O-Toulidine test where O-toulidine (aromatic amine) in hot acidic solution will yield a colored compound (due to formation of Schiff base) with an absorbance maxima at 630 nm.

Glucose in urine can be determined by Benedict’s reagent as qualitative method. Other reagent strips are also available and have glucose oxidase in chromogenic assay. These strips has impregnated with glucose oxidase, peroxidase, and the dye o-toulidine, tetramethylbenzidine. The test end of strip is moistened with freshly voided urine and examined after 10 seconds. A blue color develops if glucose is present at 100 gm/dL or more. Some strips have iodine where hydrogen peroxide produced oxidizes iodide to iodine yielding various intensities of brown color corresponding to glucose concentration in urine.

GLUCOSE METERS

A sample of blood from fingerstick but anticoagulated blood is placed on the test pad. The strip is then inserted into the meter. After a fixed period of time the result appears on digital display screen. The meter use reflectance photometry or electrochemistry to measure the rate of reaction. Reflectance photometry measures the amount of light reflected from a test pad containing reagent. In electrochemical systems, the enzymatic reaction in an electrode incorporated on the test strip produces a flow of electrons. The current which is directly proportional to the amount of glucose is converted to a digital readout. The pad contains dry GOD-POD and chromogen.

Many glucose sensors with electrochemical detectors can be implanted intravenously or subcutaneously. Some instruments can be kept on skin which senses the movement of glucose in extra and intravascular space that produces small current and this is sensed. 


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