Practical 2: Estimation of Glucose in the serum/urine sample Aims:

The aim of this experiment was to measure the concentration of glucose in the given urine and plasma samples based on glucose oxidase principle using spectrophometric technique.

Introduction:
Glucose is the monosaccharide, the simplest form of sugar and the major source of energy in our body. It is transported in the bloodstream and distributed to the body tissues. The normal fasting blood glucose level in humans lies within the range 0.1 1.1g/L or 3.9 - 6.1mmol/L. If the blood glucose level in one person is greater than 6.1mmol/L, this person could be at risk of having some defect in carbohydrate absorption or metabolism. Hyperglycemia is the term for high blood glucose level due to the insulin secretion deficiency or the insulin cannot function appropriately where the excessive glucose cannot be converted into glycogen through a process known as glycogenesis and stored in the liver or muscle cells. Sometimes psychological conditions for instance, fear and nervous may also elevate the blood glucose level. On the other hand, hypoglycemia is the condition when the blood glucose level is too low, due to the inability of the glucagon secretion, a hormone that converts the glycogen to glucose when necessary. The process of the breakdown of glycogen into glucose is called glycogenolysis .In addition, hypoglycemia may occur when the glucose is unable to be synthesized from non-carbohydrate substrates such as amino acids and fatty acids, this process is known as gluconeogenesis. Diabetes mellitus is a severe fatal disorder which is caused by the inability of glucose uptake by cells. This inability might be due to either insufficient insulin secretion or a defect in the insulin-binding receptors on cell membranes. The level of blood glucose is regulated by two major hormones insulin and glucagon, although sometimes adrenaline has certain effect. Insulin is the hormone secreted by cells of islets of Lang erhans in the pancreas that reduces the blood glucose concentration by converting the excessive 1

glucose to glycogen, the storage form of glucose in the liver or muscle cells. Therefore, in diabetes mellitus, the pancreas unable to secrete insulin causes the blood glucose level to increase dramatically, which may also lead to glucose excretion in urine. The complications of diabetes mellitus include kidney dysfunction, blindness, and lower limb amputations. The condition where glucose is present in urine is known as glycosuria. However, it is rarely seen as glucose normally will not be excreted into the urine. Hence it is important to monitor the blood glucose level regularly. Glucose oxidase method is one of the frequently used methods in clinical assay of measuring blood glucose concentration in serum samples of urine and blood plasma. It is an enzymatic method which using both glucose oxidase and peroxidase enzymes. Glucose is oxidized into gluconic acid and hydrogen peroxide is formed in the presence of glucose oxidase enzyme. The hydrogen peroxide produced reacts with phenol and 4aminophenazone in the presence of peroxidase enzyme to form a quinoneimine, a redviolet colored complex. The greater color intensity indicates the glucose concentration is high in the solution. Spectrophotometry is the quantitative measurement of the reflection or transmission properties of a material as a function of wavelength. The Beer-law is applied as the principle in spectrophotometry. The Beer-Lambert Law states that absorbance is directly proportional to the concentration (c) of the absorbing molecules, the distance travelled by light through the medium and the molar extinction coefficient: A = cl where = molar extinction coefficient for the absorbing material at wavelength in units of 1/(mol x cm) c = concentration of the absorbing solution (mol/L) l = light path in the absorbing material (l=1 cm) In practical, Beer-Law is fairly accurate for range of chromophores, solvents and concentrations, and is a commonly used in quantitative spectroscopy..Here we are using spectrophotometer to detect the absorbance of the samples at 500nm in order to determine the glucose concentration in the sample solutions. 2

Materials and apparatus:

Standard serum, Control serum C2 and C3, Serum samples A and B (Urine and plasma), Glucose oxidase reagent, Spectrophotometer, Cuvettes, Micropipettes and pipette tips

Methods:
1. Six cuvettes were prepared as table below: Contents of the tube Blank Standard Control-2 Control-3 Sample-A Sample-B Reagent Cuvette 1 Blank ------------------------------------2ml Cuvette 2 Standard ------20l ------------------------2ml Cuvette 3 Control-2 ------------20l ------------------2ml Cuvette 4 Control-3 ------------------20l ------------2ml Cuvette 5 Sample A (Urine) ------------------------20l ------2ml Cuvette 6 Sample B (Plasma) ------------------------------20l 2ml

2. Reagents were mixed appropriately. 3. The cuvettes were incubated for 25 minutes in room temperature 4. The spectrophotometer was zero with the blank and the absorbance were measured within I hour at wavelength of 500nm.

Results:
Tube Blank Standard Control 2 Control 3 Sample-A Sample-B Absorbance at 500nm 0.000 0.538 0.406 0.811 1.358 1.564 Concentration in mmol/litres ------5.43 4.10 8.19 13.71 15.79

Calculation:
The glucose concentration can be calculated using the following formula:

Concentration of glucose = Standard concentration x

Concentration of glucose in sample A = 5.43 mmol/litres x = 13.71 mmol/litres.

Concentration of glucose in sample A = 5.43 mmol/litres x = 15.79 mmol/litres.

Discussions:
First of all, we know that the normal glucose concentration is within 0.7 -1.1 in gram per litre or 3.9-6.1 in millimole per litre. The various serum samples were tested here and color formation indicates the presence of glucose in the sample solutions. The blank solution which consists of the glucose oxidase reagent is required to zero the instrument as some amount of absorbance may be taken by the reagent. Standard solution containing the standard glucose concentration of 5.43 mmol/L is used to compare with other sample solutions. The theoretical glucose concentration of Control 2 is within 5.37 - 7.27 mmol/L whereas for Control 3 is 13.1- 17.9 mmol/L. In practical, the glucose concentration in Control 2 and Control 3 solution are not in the normal ranges, which are 4.10 mmol/L and 8.19 mmol/L respectively. The error might be due to the Control 2 and Control 3 was not stored properly or they has been contaminated which causes the changes in concentration. Nevertheless, the urine sample shows an extremely high glucose concentration of 13.71 mmol/L. Therefore we assume that the sample is diagnosed as glycosuria. Glycosuria is a rare condition where the glucose is filtered and excreted in the urine. It might be due to some problem with kidneys associated with diabetes complications. As for the plasma sample, the glucose concentration is 15.79 mmol/L which is abnormally high compared to the normal one. Hence we can deduce that this patient is suffering from diabetes. A diabetic patient will have the specific conditions such as nephropathy, neuropathy, glaucoma, hyperglycemia, and more severe case may contribute to cardiovascular diseases. Besides that, several errors may occur during practical which influence the results. Firstly, the Inappropriate pipetting techniques in taking the solution will affect the results. While we holding the cuvettes, our fingerprints might be there on the cuvettes, it should be cleaned properly before measuring the absorbance, otherwise the absorbance obtained is not accurate. There is also a possibility that the solutions are contaminated during the practical. In addition, the reaction need to be completed before measurement by ensuring there is no air bubbles in the solution. The presence of air bubbles is due to the development of hydrogen peroxide after adding the reagent to the solutions. The 5

mixture should not be left for a long time, its absorbance should be measured within 1 hour after 25 minutes incubation at room temperature.

Conclusions:
The results obtained show that the concentration of glucose in urine is 13.71 mmol/L, which is extremely greater than the normal range 0.1 - 0.8 mmol/L, whereas in plasma sample the glucose concentration is 15.79 mmol/L, that is also out of the normal range 4.2 6.4 mmol/L. Thus, we can conclude that the samples tested are diagnosed as diabetes.

References:
1. Hyperglycemia. Retrived 13 March, 2013 from http://www.diabetes.org/living-with-diabetes/treatment-and-care/blood-glucosecontrol/hyperglycemia.html 2. Hypoglycemia. Retrived 13 March, 2013 from http://www.ncbi.nlm.nih.gov/pubmedhealth/PMH0001423/ 3. Beer-Lambert Law. Retrived 13 March, 2013 from http://www.oceanoptics.com/technical/beerslaw.asp 4. Murray, R. K. (2012) Harper's Illustrated Biochemistry 29 edition United States: McGraw Hill

Questions:
1. What are the preservatives used in the collection of blood glucose? The preservatives used in collection of blood glucose are D-mannose, glyceraldehyde or combination of D-mannose and sodium fluoride (NaF). 2. State the enzyme involved in this method. This method involves glucose oxidase and peroxidase enzymes. GOD Glucose + O2 + H2O 2 H2O2
+

4-aminophenazone + Phenol

gluconic acid + H2O2 POD quinoneimine + 4 H2O

3. State whether the sample A and sample B is within the specified range or not. No. The glucose concentrations for both of the samples are out of the specified range. The glucose concentration in urine sample is 13.71 mmol/L whereas in plasma sample is 15.79 mmol/L. 4. What are the advantages of GOD method than Hexokinase method? GOD method has more advantages than Hexokinase method due to its stability and GOD is much cheaper compared to Hexokinase. And it is unnecessary for GOD to keep refrigerated. 5. What are the common errors in this method? i. ii. iii. iv. v. vi. The cuvette surface might have the fingerprints on it. Inappropriate pipetting techniques in taking the solution. The solutions are contaminated, not pure The measurements were taken before the reaction is completed The presence of air bubbles in the solution The absorbance was not measured within 1 hour after 25 minutes incubation at room temperature.

6. What is the importance of glycolysis in carbohydrate metabolism? Generally glucose is converted to pyruvate through anabolic and catabolic reactions. It plays an vital role in cellular respiration .This process is important in producing energy from glucose in the form of ATP (adenosine triphosphate) and pyruvate is formed.

Glycolysis can be carried out aerobically to produce acetyl CoA or anaerobically.to produce lactate. 7. Write the pathway of TCA cycle. In the Tricarboxylic Acid (TCA) cycle, the first reaction is the conversion of pyruvate to acetyl CoA. Two-carbon acetyl group from acetyl-CoA is then transfer to four-carbon acceptor compound (oxaloacetate) to form a six-carbon compound (citrate). The citrate will then undergo a series of chemical transformations, losing two carboxyl groups as CO2. Most of the energy made available by the oxidative steps of the cycle is transferred as energy-rich electrons to NAD+, forming NADH. For each acetyl group that enters the TCA cycle, three molecules of NADH are produced. Electrons are also transferred to the electron acceptor. At the end of each cycle, the four-carbon oxaloacetate is reformed, and the cycle continues. The TCA cycle is described in details in the following diagram: