Gaines test.

Quantitative identifying of amylase activity in blood serum.

Urine and blood serum of healthy people have low amylase activity in comparison with saliva. Identifying amylase activity in urine and blood serum is used in clinical practice while diagnosing thepancreatic gland diseases. In acute pancreatitis, the enzyme activity in blood and urine is increased 10-30 times, in chronic pancreatitis, pancreatic cancer in 3-5 times. Decreased kidney function (renal disorder) as well as some blood disorders cause enzyme activity increase only in blood but not in urine.

Amylase enzyme catalyzes the hydrolysis of α-1,4-glycosidic bonds of starch and glycogen. The method is based on colorimetric estimation of starch concentration before and after enzymatic hydrolysis according to coloring in reaction with Lugol’s reagent. Do the work according to table.

Table. Identifying amylase activity.

Chemical reagents	The amount of chemical reagent, ml
experiment	control
Starch solution	1,0	1,0
Blood serum	0,02	-
	Incubate at 37^0C during 5 minutes, then add:
Lugol’s reagent	1,0	1,0
Distilled water	8,0	8,0
Blood serum	-	0,02

Photometer both of the solutions using photoelectric colorimeter at 630 nm (the thickness of the cuvette is 10 mm) against water.

The amylase activity is counted using the formula: ((D_c – D_e)/D_c)×200, where D_c is optical density of the control test, D_e – of the experiment test.

Express diagnostics of carbohydrate metabolism pathologies.

Hyper- or hypoglycemia (high or low blood glucose levels) are characteristic of some diseases. Glucose is found in very small quantities in the urine of healthy human. It cannot be found by common qualitative reactions. The phenomenon when glucose in urine is determined by common chemical methods is called glycosuria. Glycosuria may be a consequence of hyperglycemia and to be associated with decreased insulin production, hyperfunction of adrenal glands, pituitary gland, thyroid gland, as well as a one-time intake of large amounts of sugar. Therefore, the determination of glucose in biological fluids is important for the clinical diagnostics.

2.1. Trommer’s tes.

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.

Gaines test.

Add 1 ml of urine to 3-4 ml of Gaines reagent (prepared from copper sulfate, sodium hydroxide and glycerol). Heat the upper part until boiling of the reaction mixture. You can observe the appearance of yellow, then red color in the presence of glucose in urine.

2.3. 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.

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 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.

Дата добавления: 2015-10-29