Experiment 2. Quantitative determination of pyruvic acid in urine.

Pyruvic acidis 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 acidis one of the main sources for gluconeogenesis. As a result of high rate of the conversion pyruvic acidis present in tissues and biologic liquids in little amounts. In blood its amount is 0,5-1 mg/100 ml. The amount of pyruvate in urine is normally 2 mg/100 ml, its 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 during muscular work and В₁ vitamin deficiency. This phenomenon is also noticed during 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 quantity of pyruvic acid.

pyruvic acid 2,4-dinitrophenylhydrazine dinitrophenylhydrazone of pyruvic acid

Course of work: Pour 0,5 ml of 0,1% 2,4- dinitrophenylhydrazine up to 0,5 ml of urine, mix and add 2,5 ml of water-saturated toluene after 5 minutes. After shaking for 1 minute leave the solution for water and toluene demixing. Take a 1 ml test from the upper toluene layer with a dry graduated pipette and place it into a dry test tube, add 3 ml of potassium hydroxide alcohol solution. Process the test with 1 ml of standard solution of pyruvate in the same way as the experiment test. In the chemical reagents control the urine is substituted by 1 ml of water. After 10 minutes photometer the tests against the chemical reagents control with the wave length of 400-415 nm using photoelectric colorimeter.

The calculation is made according to the formula: Х=(D_e*50mg)/D_s, where: D_e – is optical density of the experimental solution, D_s – is optical density of the standard solution, 50 mg – is the concentration of pyruvic acid standard, Х – is the concentration of pyruvate in urine.

Test questions

1. What reactions catalyze the enzymes of oxidoreductases class?

2. Consider the role of dehydrogenases in the processes of biological oxidation.

3. For experiment 1 make up a scheme of hydrogen transfer (electrons and ions of hydrogen) taking into account standard oxidation-reduction potentials of the used components.