Experience 2. Express diagnostics of carbohydrate metabolism pathologies.

2.1. Trommer’s test with copper hydroxide.

Pour 1 ml of 10% sodium hydroxide solution up to 1 ml of urine and 1% copper sulphate solution drop by drop till then on sediment of copper hydroxide (II) appears. The appearance of red coloring when heated testifies about the presence of glucose in urine.

2.2. Discovery of fructosuria by Selivanov’s test.

The method is based on the conversion of fructose when heated and in the presence of hydrochloric acid into hydroxymethyl furfural which condenses with resorcin (0,05% solution in 20% hydrochloric acid, Selivanov’s reagent), forming the compound of red color.

Pour 1 ml of resorcin solution into 2 test tubes. Add 2 ml of normal urine to the one of them, and the same amount of pathological one to the second test tube. Heat both the test tubes in the water bath till boiling. If fructose is present in the urine there will be bright red coloring. It should be checked at the first moment of boiling; otherwise longer time heating can make glucose give the positive reaction.

2.3. 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 and 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 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 2 minutes. Compare the changed coloring of the colored 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.

References

1. Berezov, Т.Т. Biological chemistry. / Т.Т. Berezov, B.Ph. Korovkin. - М.: Open joint-stock society ""Medicine"publishing office", 2007. - 704 p.

2. Biochemistry. / Edited by Y.S. Severin. - М.: ГЭОТАР-Меdia, 2003. - 779 p.

3. Biochemistry. The short course with exercises and tasks. / Edited byY.S. Severin, А.Y. Nikolayev. - М.: ГЭОТАР- Меdia, 2001. - 448 p.

4. Zubairov, D.М. Study guide for laboratory works on biological chemistry. / D.M. Zuibarov, V.N. Timerbayev, V.S. Davydov. - М.:ГЭОТАР-Меdia, 2005. - 392 p.

5. Kushmanova, O.B. Study guide for laboratory works on biological chemistry. / О.B. Kushmanova, G.I. Ivchenko.- М. - 1983.

6. Lenindger, А. The fundamentals of biochemistry / А. Lenindger. - М., «Mir». - 1985.

7. Murry, R. Biochemistry of a man. / R. Мurry, D. Grenner, P. Meyes, V. Rodwell. - V. 1. - М.: Mir, 1993. - 384 p.

8. Murry, R. Biochemistry of a man. / R. Мurry, D. Grenner, P. Meyes, V. Rodwell. - V. 2. - М.: Mir, 1993. - 415 p.

9. Nikolayev, A.Y. Biological chemistry. / А.Y. Nikolayev. - М.:Medical information agency, 2004. - 566 p.

10. Fundamentals of Analytical Chemistry. / Ed. J.A. Zolotov. - Book 2. - M.: High School, 1996. – 461 p.

11. Reference book. Laboratory methods of investigation in clinic. / Edited by prof. V.V. Menshikov. - М. Medicine. - 1987.

12. Philippovich, Y.B. Fundamentals of biochemistry. / Y.B. Philippovich. - М.: High school, 1993. - 496 p.

13. Philippovich, Y.B. Exercises and tasks on biological chemistry. / Y.B. Philippovich, G.А. Sevastyanova, L.I. Schegoleva. - М.:Prosveschenie, 1986. - 144 p.

14. Philippovich, Y.B. Practicum on general biochemistry / Y.B. Philippovich and others.–Textbook. - М.:Prosveschenie, 1982.

15. ElliotV., ElliotD. Biochemistry and molecular biology. / V. Elliot, D. Elliot. - М. - 1999