Laboratory work 8. Carbohydrate metabolism

Food substances (starch, glucose, fructose, sucrose, lactose, maltose) are carbohydrate source of organism. Starch is the storage form of glucose in plants; lactose is found in milk, glucose and fructose - in fruits and honey, maltose is present in products that contains partially hydrolyzed starch, such as malt. Normal intake of carbohydrates is 400-500 g/day. Carbohydrates are the main source of energy for human body.

Polysaccharides and disaccharides are digested in the gastrointestinal tract. Starch is partially digested in the mouth by α-amylase of saliva, splitting α-1,4- glycosidic bonds. Dextrin and maltose are formed. Pancreatic amylase hydrolyzes starch in the upper part of the small intestine. Maltose and isomaltose are formed. Maltose, isomaltose, sucrose and lactose are hydrolyzed by glycosidases on the surface of cells of the small intestine to form monomers. The resulting monosaccharides are absorbed in the intestine, enter the bloodstream and then come to the liver through the portal vein. Part of glucose passing through the liver, is carried by the blood throughout the body, but the main part is deposited in the liver as glycogen. When blood glucose level is decreased, glycogen is broken down to glucose. Despite continuous glucose consumption by all cells in the body its concentration in the blood is maintained at a steady level 3.3-6.1 mmol/l. Constancy of the blood glucose concentration is regulated by the action on the liver through the central nervous system and the endocrine system (mainly - pancreas, pituitary and adrenal glands).

Hyper- or hypoglycemia (high or low blood glucose levels) are characteristic of some diseases. Glucose is found in very small quantities in 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 clinical diagnostics.

1. 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 the pancreatic gland diseases. Normally alpha-amylase activity in blood is 28-100 IU/L, pancreatic amylase activity – 0-50 IU/L. Alpha-amylase activity in urine is 1-17 IU/h. 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 the Table.

Table. Identifying amylase activity.

Chemical reagents	Volume, ml
	Test tube 1 - experimental	Test tube 2 - control
Starch solution	1,0	1,0
Blood serum	0,02	
	Incubate at 370 C for 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.

2. Express diagnostics of carbohydrate metabolism pathologies.

All experiments should be done with urine samples 1 and 2.

2.1. Trommer’s tes.

Pour to 1 ml of urine in the test tube and add 1 ml of 10% sodium hydroxide solution. Then add 1% copper sulfate solution drop by drop till the precipitate of copper hydroxide (II) appears. The appearance of red coloring when heated testifies about the presence of glucose in urine.

2.2. Gaines test.

Pour 3-4 ml of Gaines reagent (prepared from copper sulfate, sodium hydroxide and glycerol) in the test tube and add 1 ml of urine. Heat the upper part of the solution until boiling of the reaction mixture.

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 resorcinol (0.05% solution in 20% hydrochloric acid, Selivanov’s reagent), forming the compound of red color.

Pour 1 ml of resorcinol solution into 2 test tubes. Add 2 ml of urine and heat the test tube in the water bath till boiling. If fructose is present in the urine there will be bright red coloring.