PRACTICE 4. CARBOHYDRATES

Guidelines1. Read carefully a theoretical background in the introductory part of practice.2. Remind on classification of carbohydrates according to their structural complexity.3. Recognize specific structural features of carbohydrates focused to different stereoisomers, types of glycosidic bonds, reducing and non-

reducing properties of sugars.4. Review your knowledge on degradation of dietary sugars in digestive system, with particular emphasis on localization of digestion,

localization of different digestive enzymes, and their actions and main products of dietary sugars digestion.5. Review your knowledge on major types of glucose utilization in carbohydrate energy metabolism.

Exercises1. Detection of salivary α-amylase

Please read carefully the principle of two reactions: with iodine, for detecting starch; Trommer’s reaction for determination of reducing properties of monosaccharides.

Answer three questions listed within exercise 1.2. Determination of glucose concentration in blood

Read the principle of the method Use given data for calculation of concentration of glucose in your sample, and briefly comment the result.

3. Review questions Answer Review questions, except the Q3. Your answers should be concise; use only words or simple schemes (no structural

formulas are needed in your replies).

Several next slides are reviewing major important data about sugars, needed to understand and correctly solve your exercises/questions.

Svjetlana Kalanj Bognar (svjetlana.kalanj.bognar@mef.hr) and Tamara Božina (tamara.bozina@mef.hr)

are polyhydroxy aldehydes and polyhydroxy ketones

MONOSACCHARIDES

Cyclic forms are more stable structures of a monosaccharides.

In natural sources, glucose is found predominantly incyclic pyranose form and fructose in cyclic furanose form.

Enantiomers – the opposite configuration at every chiral center.Glucose and all other naturally occurring monosaccharides (sugars) are D-sugars.

D-glucose L-glucose

D- and L-sugars

Diastereomers differing in the configuration around one stereogenic center only are called epimers.

D-mannose D-glucose D-galactose

Formation of glycosidic bonds requires involvement of at least one (the most reactive) hemiacetal –OH group with theoretically any of the alcoholic functional group of another monosaccharide.

Oxidation is one of a typical reactions of monosaccharides

Monosaccharides can be oxidized by relatively mild oxidizing agents (Fe3+ and Cu2+) –qualitative methods for determination of presence of a reducing sugars are based on thisproperty of monosaccharides (Trommer’s reaction); enzymatic method is used in laboratorypractice (glucose oxidase method).

All monosaccharides are reducing agents!

The carbonylgroup is oxidized

to carboxyl group.

Ketoses act as reducing sugarsdue to isomerisation reaction involving formation of enediol intermediate under alkaline conditions

– Trommer’s reaction is positive in solution containing fructose.

All monosaccharides are reducing agents!

Maltose type disaccharides

REDUCING DISACCHARIDES-Maltose-Lactose(free reactivehemiacetal hydroxy groups!)

-Trehalose-Sucrose(hemiacetal hydroxy groups from bothmonosaccharides are involved in formation ofglycosidic bond!)

NONREDUCING DISACCHARIDES

Trehalose type disaccharides

In general, polysaccharides do not show reducing properties!

STARCH

GLYCOGEN

CELLULOSE

Principle of reaction of starch with elementary iodine: In reaction with elementary iodine, starchgives the intensively dark blue non-covalent product. Namely, the iodine molecules incorporateinto cavities inside the coil of starch polysaccharide chain; due to high light absorption, theresulting so-called adsorptive compound shows characteristic dark blue color. As α-amylasedegrades starch, the color of the adsorptive starch-I2 compound disappears due to decay of itsstructure.

Principle of Trommers’s reaction (Fehling’s test): This test is used for determination of reducingproperties of monosaccharides. The Fehling’s reagent is very alkaline solution of complexcopper(II) tartrate. This is the reason why in this reaction other reducing sugars or other reducingsubstances, besides glucose, are easily oxidized.

EXERCISE 1.Detection of salivary α-amylase

Your first task will be to conclude on the effect of boiling the saliva sample on activity of salivary α-amylase inreaction mixture containing saliva, starch and Lugol solution. Comment what you expect as results ofreactions in test tube 1 (containing not-boiled saliva) and test tube 2 (containing boiled saliva).

Your second task will be to predict the result of performing Trommer’s reaction in test tube 1 and test tube 2.

EXERCISE 2. Determination of glucose concentration in blood

Principle of the enzymatic and spectrophotometric method for determination of glucose concentration: Glucose-oxidase (GOD)catalyzes the oxidation of glucose to gluconic acid yielding the H2O2. In a peroxidase (POD) catalyzed reaction, the formed H2O2oxidizes the colorless chromogen into a colored compound. The intensity of color is directly proportional to the glucoseconcentration in the sample.

Reference values: serum, plasma: 3.9 – 5.8 mmol/L

c(glucose) = ___________mmol/L

A st 0.35

A sm 0.32 (group 1) 0.15 (group 2) 0.73 (group 3)

c st/mmol L-1 5.55

Your task will be to calculate concentration of glucose in your sample. For calculation you will use: 1. given value for absorbance measured in a standard solution (A st)2. given value for absorbance measured in your sample (A sm)3. known concentration of standard glucose solution (c st)

You will write down your result, compare it with reference values, and briefly comment whether the obtained glucose concentration is within reference range or not (possible results will be: normoglycemia, hyperglycemia and hypoglycemia).

Note: For calculating glucose concentration, all students use the same value for A st and c st. For values of the A sm, students are divided in three groups, and should use the value for a specified group, as explained below the table.

Group 1 – Bekavac, Ghoddoosi, Marcelić, Petranović, Sudarević, Tepšić, Verreault Group 2 - Eilo, Herman, Markusi, Mnushkin, Srejić, Tittmann, Vranić, ŠramelGroup 3 - Filipović-Grčić, Ivančić, Mikho, Rudolf, Šegan, Trampuš, Žlebnik