TASK 1 BIOCHEMISTRY

determination of glucose
WRITTEN BY : IKA YULIA RIZKI 1111012053

Lecturer : Najmiatul Fitria, M.Farm, Apt

PHARMACY FACULTY ANDALAS UNIVERSITY PADANG 2012

DETERMINATION OF GLUCOSE
The word carbohydrate arose because the molecular formula of this compound can be expressed as a hydrate of carbon. For example, glucose has the formula C6H12O6 that can be written as the C6 (H2O) 6. Carbohydrates are polihidroksialdehid, polihidroksiketon, or substance that gives such compounds when hydrolyzed. Carbohydrate chemistry is basically a chemical combination of two functional groups, ie hydroxyl and carbonyl groups .Carbohydrates or saccharides are the major class of organic compounds composed of carbon, hydrogen, and oxygen (Ratna et al., 2010).

The simplest form of carbohydrate molecules consisting of a simple sugar molecule. If the carbon atom is denoted as a black ball, okeigen red and white, then the hydrogen molecule three-dimensional shape of the glucose would like the picture beside it. Many carbohydrates that are polymers composed of sugar molecules that are strung into a long chain and branched.

Carbohydrates are an important food and source of energy contained in plant and animal flesh. In addition, carbohydrate is also an important structural component of living things in the form of fiber (fiber), such as cellulose, pectin, and lignin (Ratna et al., 2010). Carbohydrate present in all plants and animals and are essential for life. Through photosynthesis, plants convert atmospheric carbon dioxide into carbohydrates, mainly cellulose, starch, and sugar. Cellulose is the building block on a rigid cell wall and woody tissue in plants, while starch is the main form of carbohydrate reserve for later use as food or energy source. Some plants (cane and beet sugar) sucrose yield. Other sugars, namely glucose, an important component in the blood. Two other sugars, ribose and 2-deoxyribose, is a component of genetic material RNA and DNA. Other carbohydrates important as a component of coenzymes, antibiotics, cartilage, shells of crustaceans, bacteria cell walls and membranes of mammalian cells (Hart et al., 2003).

Simple carbohydrates can be viewed as polyhydroxy aldehydes and ketones. The simplest carbohydrates are monosaccharides. When the sugar has an aldehyde group, the sugar is an aldose. However, when the sugar has a keto group, the sugar is a ketosa. A monosaccharide recognition of number of carbon atoms they contain. Monosaccharides are most often found in our food are the hexoses glucose and fructose. Based on the number of monomers forming a carbohydrate, it can be divided into three major categories namely monosaccharides, disaccharides and polysaccharides. The term saccharide derived from the Latin and refers to the sweetness of simple carbohydrate compounds. Monosaccharides are carbohydrates that can not be hydrolyzed into simpler compounds (Tim Lecturer of Chemistry, 2008).

Monosaccharides are simple carbohydrates, in the sense that the molecule consists of only a few carbon atoms and can not be described by way of hydrolysis in a soft condition of the other carbohydrates. As for some important monosaccharides namely glucose, fructose, galactose and pentose. Glucose is a aldoheksosa and often called dextrose because it has the
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properties of polarized light to rotate to the right. In nature, the glucose found in fruits and honey bees. In the nature of glucose produced from the reaction between carbon dioxide and water with the help of sunlight and chlorophyll in the leaves. This process is called photosynthesis and produce glucose which is used for the formation of starch and cellulose (Poedjiadi, 1994).

Glucose is a sugar having six carbon atoms and thus are called hexoses. Five-carbon carbohydrate known as the pentose and the next. The fact that an aldehyde carbonyl group was demonstrated by characterizing glucose as aldoheksosa. Very important monosaccharide D-glucose is often known as dextrose. Open form of hexoses are in equilibrium with a form or hemiketalnya hemiasetal. Glucose has the most dominant form of piranosa and second anomer and have been isolated. Based on the definition, which has a shape isomer C1-OH and C5-C6. Cyclization will generate a new asymmetric center. Anomer different in physical properties and optical rotation. In both forms of solution to reach equilibrium and the reaction can be followed by measuring the change in optical rotation. These changes are called mutarotasi (Sastrohamidjojo, 1996).

CHO H HO H H OH H OH OH CH2OH

D glucose

Fischer projection formula is a common way to describe the monosaccharide molecules. Ordinary projections were drawn with a vertical carbon chain and the carbonyl group closest to the peak. Reducing sugar is sugar that has the ability to reduce. This is because
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the presence of free aldehyde or ketone group. Compounds are oxidizing or reducing agent is an oxidizing metals such as Cu (II) (Budiyanto, 2002).

Glucose can be reduced ion kupri kupro that this reaction can be used as a basis in the determination of glucose and performed by various methods, among others: Luff Schrool, Munson-Walker, Lane-Eynon and Somogy-Nelson. Somogy-Nelson method is based on the reduction of ion kupri by glucose (sugar reduction) under alkaline conditions with arsenomolibdat which gives a blue color. The intensity of color formed depends on the glucose concentration (Tim Lecturer of Biochemistry, 2010).

Nelson tried various color reagents, which led to the development of a new reagent arsenomolybdate. When the reagent is used by Somogyi micro reagent, it gives satisfactory stability and reproducibility of color. By this means has been possible to use copper reagents in photometric procedure for almost all used for titrimetric procedures are adapted. These include tissue sugar, glycogen, urine reducing equivalents, maltose, glucuronic acid, etc. (Nelson, 1944).

Determination
fasting blood is sampled for glucose determination. the concentration of glucose can be determined in whole capillary or venous blood, serum or plasma. in whole blood, glucose can only be determined by manual methods, whereas determination of glucose in serum or plasma can be performed by automated methods. the concentration of glucose in whole blood is by 10% to 15% higher than in serum or plasma. for methodologic (some erythrocyte substances react in particular analytical methods) and physiologic reasons (glucose concentration in plasma is a physiologic stimulant for insulin action), glucose should preferably be determined in serum or plasma rather than in whole blood. in whole blood, glucose is degraded by the glycolysis enzymes from erythrocytes and
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leukocytes. decrease in the sample concentration of glucose following blood sampling should be prevented by one of the following procedures: blood sample deproteinization, blood sampling into a tube containing an anticoagulant inactivating glycolysis enzmyes [the most commonly used anticoagulants are sodium fluoride (1 mg/ml blood), sodium oxalate, or potassium edta (1 mg/ml blood)], or separation of blood fluid from blood cells after centrifugation.

determination of glucose in whole blood, serum or plasma is one the basic tests in daily laboratory routine. numerous methods nonspecific for glucose used to be employed, however, the specificity has been greatly improved with the introduction of enzymatic methods, therefore they should be given preference to other methods. the hexokinase method is currently considered the reference method for determination of glucose concentration. besides specificity, this method has a number of other advantages: it is sensitive, accurate and rapid, and covers a wide range of glucose concentration in the sample. enzymatic methods for glucose determination are classified into three groups: 1. methods with glucose oxidase, 2. methods with hexokinase, and 3. methods with glucose dehydrogenase.

Glucose Oxidase Methods

the principle of these methods is based on glucose oxidation to gluconate by the action of glucose oxidase enzymes (god), and formation of hydrogen peroxide. this step of reaction is specific for glucose. in the next, indicator reaction, the utilization of oxygen is measured or the reaction is based on the hydrogen peroxide formed, which oxidizes a chromogenic substance to a colored product under the action of peroxidase (pod). the intensity of absorbance of thus formed stained product is proportional to the concentration of glucose in the sample. various chromogenic substances can be used, e.g.; 1. o-dianizidine
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2. abts (2,2''-azino-di-3-ethylbenzothiazoline sulfonic acid 6-diammonium salt) 3. 4-aminoantipyrine dimethylanilline 4. 4-aminoantipyrine abts and 4-aminoantipyrine are most commonly used as a chromogen. accordingly, there are two methods: 1) god-perid method, and 2) god-pap method.

God-Perid Method
the specific enzyme glucose oxidase (god) oxidizes glucose with the formation of h 2o2. thus formed h2o2 oxidizes abts chromogen into a green-stained compound. the absorbance of oxidized, green-stained chromogen is measured photometrically at 436 nm or between 560 and 660 nm. test strips for quantitative determination of blood glucose concentration are made on the principle of god-perid method. they are used for fast differentiation of glycemia.

God-Pap Method
in the presence of peroxidase, hydrogen peroxide reacts with 4-aminoantipyrine to form a redstained compound with a maximal absorbance at 510 nm. the 4-aminoantipyrine chromogen is stable and resistant to auto-oxidation, therefore the method is highly reliable and specific for glucose.

Hexokinase Method
the hexokinase method is a reference method for determination of glucose concentration. the method is specific for d-glucose.

under the action of the hexokinase enzyme, d-glucose is phosphorylated with atp molecule to form glucose-6-phosphate. by the action of glucose-6-phosphate dehydrogenase (g-6-pdh) in the presence of nadp, thus formed glucose-6-phosphate transforms to 6-phosphogluconate,
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whereby nadph is formed. the absorbance of nadph is measured in the uv region (334, 340 or 365 nm). besides glucose, fructose and mannose can also react in primary reaction. however, g-6-pdh is specific exclusively for glucose-6-phosphate, thus phosphorylated fructose and mannose do not react in indicator reaction.

Blood Glucose Monitoring Systems Are Based On A Reflectometric Hexokinase Method

Glucose Dehydrogenase Method

the principle of reaction is as follows: by the action of glucose dehydrogenase enzyme (gdh) with nad, d-glucose and nad are transformed to d-gluconolactone and nadh. the change in nadh absorbance is measured in the uv region (334, 340 or 365 nm), because the amount of the reduced coenzyme formed is proportional to the concentration of glucose.

the hemocue glucose systems are based on a glucose dehydrogenase method

Experiment Methodology
Tools
The tools used in this experiment is spektronik 20 D +, digital analytical balance, 10 mL volumetric flask, 0.2 mL measuring pipette, measuring pipette 1 mL, 10 mL measuring pipettes, pipette 1 mL volume, bulp, filler, micro pipettes , 100 mL beaker, horn spoon, stir bar, test tubes, test tube rack, pipette drops, tube clamp (tweezers), bath water, the cuvette, oven, brush tube.

Materials
The materials used in these experiments is the color reagent arsenomolibdat, Nelson solution A, solution B Nelson, glucose monohydrate (C6H1206.H2O), the sample liquid (drinks M150), distilled water, label paper, soap and tissue roll .

Work Procedures
Preparation of Parent Solution 1 mg / mL Weighed 0.011 g of glucose monohydrate, and then inserted into a 100 mL beaker and dissolved with a little distilled water and then put into 10 mL volumetric flask and diluted with distilled water until the volume reached 10 mL. Then the homogenized solution.

Preparation of Standard Solution Mother liquor pipetted into 5 pieces as much as 0.02 mL test tube at first tube, 0.03 mL in the second tube, 0.04 mL, 0.05 mL in the third tube in the fourth tube and 0.06 mL on the fifth
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successive tubes -contribute to making a standard solution with a concentration of 0.004 mg / mL; 0.006 mg / mL; 0.008 mg / mL; 0.010 mg / mL; 0.012 mg / mL. After it was diluted with distilled water into each test tube until it reaches the volume of 5 mL. Then the homogenized solution.

Preparation of reagents Nelson Pipetted Nelson A solution of 20 mL into a test tube was added 0.8 ml of Nelson B. Then the homogenized solution.

Preparation of Sample Solution Pipetted as much as 0.1 mL of sample solution into a test tube. Then added with distilled water until the volume reached 10 mL. Then the homogenized solution. After that, as much as 0.01 mL pipetted again, from the first dilution, into a test tube. Then added with distilled water up to 1 mL volume. Then the homogenized solution. This sample solution is 10 000 times dilution.

Determination of Glucose Levels Pipetted 1 mL of each standard solution into 5 pieces of tube. Then all five test tubes containing 1 mL of standard solutions with concentrations of respectively 0.004 mg / mL; 0.006 mg / mL; 0.008 mg / mL; 0.010 mg / mL; 0.012 mg / mL and the test tube containing 1 mL of sample solution and 1 mL of blank (distilled water) was added to each solution of Nelson as much as 1 mL. Then put in a water bath for 20 minutes, then cooled immediately in cold water. After cooling, each tube was added 1 mL of reagent arsenomolibdat then every standard series, and blank samples were diluted with distilled water until a volume of 100 mL of distilled water and homogenized and then inserted back into the test tube. After that, the measured absorbance with spektronik 20 D +.

Reference
Barker, Kathy. 1998. At The Bench, A Laboratory Navigator. Cold Spring Harbor Laboratory Press, New York. Nelson, N., 1944, A Photometric Adaptation of The Somogyi Method For The Determination of Glucose, Journal of Biological Chemistry, 163, 375-380. Beishir, Lois. 1991. Microbiology in Practice : A Self Instructional Laboratory Course. Harper Collins Publisher Inc., New York

Sudarmadji, s., haryono, b., dan suhardi, 2003, Analisa Bahan Makanan Dan Pertanian, liberty Yogyakarta Universitas Gadjah Mada, Yogyakarta. Tim dosen biokimia, 2010, Penuntun Dan Laporan Praktikum Biokimia, Laboratorium Biokimia Fakultas Matematika Dan Ilmu Pengetahuan Alam Universitas Hasanuddin, Makassar. Poedjiadi, a., 2005, Dasar-Dasar Biokimia Edisi Revisi, ui-press, jakarta.
Kunst, A.; Draeger, B.; Zeigenhorn, J. In UV-Methods with Hexokinase and Glucose-6-Phosphate Dehydrogenase; Bergmeyer, H. U., Ed.; Methods of. Enzymatic Analysis, 3rd. Ed.; 1984; pp 163172. http://www.chronolab.com/point-ofare/index.php?option=com_content&view=article&id=291&Itemid=45

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