2.4. Enzymatic method of semi-quantitative identification of glucose in urine with the help of "glucophan" test strip.
"GLUCOPHAN" test strip has one reagent pad of light-yellow color, soaked with the solutions of enzymes glucose oxidase, peroxidase and coloring agent (benzidine derivative) solution. Glucose oxydase is a flavoprotein, the prosthetic group of which is FAD. It catalyzes the transfer of two atoms of hydrogen from glucose to aerial oxygen. The formed hydrogen peroxide is degraded by peroxidase enzyme, due to which the coloring agent is oxidizes. The change of the coloring agent color when it oxidizes testifies about the presence of glucose in the urine. This method allows us to identify the concentration of glucose in urine approximately from 0.1 up to 2%.
Pour a little amount of experimental urine into a glass. Dip "GLUCOPHAN" test strip into the experimental urine to soak the yellow reagent pad completely. Immediately remove the strip from the liquid, wait for 1 minute. Compare the color of the reagent pad with the colored scale. If glucose is absent in the urine the reagent pad color does not change. Find the most matching colors on the reagent pad and on the colored scale to identify the concentration of glucose approximately.
Test Questions
1. What are the optimal conditions for pancreatic amylase functioning?
2. What products will be formed from starch in the presence of pancreatic juice (in vitro)?
3. How can the presence of starch conversion products be identified in a sample?
4. What functional group of glucose molecule causes Trommer’s positive test?
5. What can be cause of hyperglycemia?
6. What diseases or physical conditions cause glucosuria?
7. What are causes of fructosuria?
Laboratory work 9. Intermediate products of carbohydrate metabolism
1. Quantification of pyruvic acid in urine
Pyruvic acid is one of the intermediate products of carbohydrate metabolism. Under anaerobic conditions (hypoxia) it is reduced into lactic acid, and under aerobic ones it undergoes oxidative decarboxylation and is converted into acetyl-coenzyme A. Pyruvic acid is one of the main sources for gluconeogenesis. As a result of high rate of the conversion pyruvic acid is present in tissues and biological fluids in little amounts. In blood its concentration is 0.5-1 mg/100 ml. The concentration of pyruvate in urine is normally 2 mg/100 ml. It daily excretion with urine is 10-25 mg.
The most rapid increase of pyruvate concentration in blood and as a consequence in urine is noticed after muscular work and in vitamin В1 deficiency. This phenomenon is also noticed in hepatic disorders, diabetes, cardiac decompensation, toxicosis, etc.
The method principle: pyruvate and 2,4-dinitrophenylhydrazine form a colored compound 2,4-dinitrophenylhydrazone of pyruvate. It is extracted from the reaction mixture by toluene. When alkali alcohol solution is added, it gradually turns red-orange; the optical density of the solution is directly proportional to the concentration of pyruvic acid.
pyruvic acid 2,4-dinitrophenylhydrazine dinitrophenylhydrazone of pyruvic acid
Take 3 test tubes. Pour 1 ml of urine diluted 3 times into the one tube (experimental sample), 1 ml of pyruvic acid standard solution into the second tube (standard sample), and 1 ml of water into the third tube (control sample).
Add 0,5 ml of 0,1% 2,4- dinitrophenylhydrazine solution to all samples, mix. After 5 min add 2,5 ml of water-saturated toluene into each test tube. After shaking for 1 minute leave the solutions for water and toluene demixing.
Take a 1 ml sample from the upper toluene layer with a dry graduated pipette from each test tube and place it into dry test tubes, add 3 ml of potassium hydroxide alcohol solution. Wait for 10 minutes.
Use the control sample for photoelectric colorimeter calibration. Measure the optical density of the standard sample and experimental sample by the wave length of 400-415 nm using photoelectric colorimeter.
The calculation is made according to the formula:
Х = De*50*3/Ds, where:
Х is the concentration of pyruvate in urine, mg|100 ml
De is optical density of the experimental sample,
Ds is optical density of the standard sample,
50 is the concentration of pyruvic acid standard solution, mg|100 ml,
3 – degree of urine dilution.