Biochemistry Write-up

Experiment 11: Determination of Sugar in Blood

NAME: JETHRO MAWIRE

REG NUMBER: R225676Q

YEAR OF STUDY: 2023 BMS 1.2

Title:

Determination Of Concentration Of Sugar In Blood By Comparing The Absorbance Values

With Those Obtained From Standard Solutions.

Monosaccharides, or simple sugars, consist of a single polyhydroxy aldehyde or ketone unit.

D-glucose is the most abundant monosaccharide in nature and it is a six-carbon sugar. It is

sometimes referred to as dextrose. Monosaccharides of more than four carbons tend to have

cyclic structures (Nelson and Cox, 2004). Glucose is formed from carbon dioxide and water

in plants through photosynthesis. Animals can synthesize carbohydrate from lipid glycerol

and amino-acids though most animal carbohydrates are derived from plants (Murray et al,

2003). These carbohydrates can be broken down to glucose.

All sugars can ultimately be converted to glucose. The glucose is broken down in a process

called the glycolytic pathway to provide energy and other intermediates for other metabolic

pathways. This process takes place in all tissues. The end product of glycolysis is pyruvate,

which still contains some potential energy, can be extracted by oxidative reactions in the

citric acid cycle and oxidative phosphorylation. The breakdown of glucose to release energy

takes place in the absence of oxygen (anaerobically) in cells deprived of oxygen resulting in

the continued production of ATP in these cells (Harvey et al, 2006). Glucose is the preferred

energy source for the brain, mature erythrocytes and active muscle. It is a major constituent

in several complex biological molecules such as starch, glycogen and cellulose.

Glucose is a white crystalline solid which is soluble in polar solvents. The normal fasting

level of plasma glucose in peripheral venous blood is 70-110 mg/Dl. In arterial blood the

plasma glucose level is 15-30 mg/dl higher than in venous blood. Soon after a meal the

values of blood glucose may rise to 120mg/dl or even higher however the glucose level soon

returns to normal fasting glucose. The glucose levels are maintained at normal fasting level

either by removal of excess glucose from the blood by the liver and the storage of it as
glycogen or by pathways such as glycogenolysis and gluconeogenesis that generate glucose

when glucose levels fall (Sackhalm and Lehman,1981).

Maintenance of a constant blood glucose level is crucial for normal bodily function. The

liver and kidney are the main organs involved in this regulation. Its regulation occurs initially

through changes in its uptake and phosphorylation to glucose-6-phosphate. Glucose uptake is

mediated by GLUT transporters followed by phosphorylation of hexose kinase. There are

also hormones that regulate the blood glucose level which are mainly insulin and glucagon.

Insulin is released when the blood glucose level is too high and causes its drop by promoting

glucose uptake by actively respiring cells and conversion of glucose to glycogen. Glucagon

increases blood glucose levels by stimulating glucose production by glycogenolysis and

gluconeogenesis. It does not affect muscle. Both insulin and glucagon are produced by cells

in the Islets of Langerhans in the pancreas (Devlin, 2010).

In some individuals regulation of blood glucose levels does not happen properly which

causes diabetes. There are two types of diabetes; Type 1 and Type 2. Type 1 diabetes comes

about due to an absolute deficiency of insulin caused by an autonomic attack on B cells of the

pancreas. Its onset is usually during childhood or puberty and its symptoms develop rapidly.

Type 2 diabetes develops gradually. It is more common. It is caused by insulin resistance and

B cell malfunction and is associated with obesity. This type of diabetes can usually be treated

by weight loss, a corrected diet and exercise (Barrette et al, 2012). If blood glucose drops to

half its normal concentration, the individual will experience confusion and if it drops fivefold

it may result in a comma or death. It is therefore very important maintaining a constant blood

glucose concentration.

The objective of this experiment was to determine the blood glucose concentration of a given

sample of blood. This was done using the Folin and Wu method.
Method:

Deproteinised samples were used for spectrophotometric analysis. The supernatant was

retained and prepared for spectrophotometric analysis by mixing with Nelson’s solution then

diluting them. Standard solutions were prepared and absorbance readings for the standard

solutions and samples were taken using a “Spectronic 20 Genesis”.

Results

Table 1: Absorbance Readings For The Different Standard Solutions

Tube Volume Of glucose Concentration After Absorbance

Standard /ml

Blank 0.0 0.0000 0.000

Standard 1 0.5 0.0125 0.063

Standard 2 1.0 0.0250 0.166

Standard 3 1.5 0.0375 0.190

Standard 4 2.0 0.0500 0.246

Table 2: Absorbance Readings For The Blood Samples

Sample Absorbance

Sample A 0.306

Sample B 0.310

Calculation for concentrations of glucose in Table 1;

C1V1 = C2V2

0.05 x 0.5 = C2 x 2

Hence; C2 = 0.0125

From Graph 1;

Absorbance for sample A = 0.306

Hence concentration in sample A = 0.0515 mg/0.2ml

Concentration in 1ml = 5 x 0.0515mg/0.2ml

= 1.288mg/ml

Therefore in 100ml = 1.288 x 100

= 129 mg/100ml

Absorbance for sample B = 0.310

Hence concentration in sample B = 0.052mg/0.2ml

Therefore in 100ml = 130mg/100ml

Concentration in sample using formula;

Glucose concentration A = 100 x (absorbance of sample/Absorbance of glucose standard)

= 100 x (0.306/0.166)

= 184.3 mg/100ml

Or = 100 x (0.310/0.166)
 = 187mg/100ml

Chemical equations;

Glucose + Cu2+ Cu+ + Gluconic Acid (conditions; OH- and heat)

2Cu+ + MO3+ 2Cu2+ + MO4+

Discussion:

The graph, Graph 1, plotted using the standard solutions had a best-fit line that was linear.

This was the expected result since absorbance was directly proportional to concentration of

glucose. This is because of the reducing properties of glucose. The greater the concentration

of glucose means it has a greater reducing ability. Cu2+ ions were reduced to give Cu+ ions.

These reduced ions combined with certain substances that form complexes that have colour.

This means that the greater the concentration of glucose was the deeper the colour; which

meant absorbance was directly proportional to glucose concentration in the standard

solutions.

The absorbance value for Sample A was 0.306, which was equivalent to the absorbance of a

solution with a concentration of 0.0515 mg/0.2ml. This was worked back to give a

concentration of 129mg/100ml. Sample B was treated in the same way and it gave a

concentration of 130mg/10oml. These results were out of the fasting blood glucose range,

which is 65-95mg glucose/100ml, but they are still acceptable since the blood glucose level

fluctuates depending on whether or not the subject from which the sample was taken had a

meal or was in the early starving state.

The experiment had certain limitations that could have affected the results. One of these is

that the colour formed fades. This is because of the re-oxidation of copper. Other errors could

have been due to errors in dilution, centrifugation and diluting.

The more reliable concentration of the samples was 130mg/100ml.

Answers to questions:

From Graph 1;

Absorbance for sample A = 0.306

Hence concentration in sample A = 0.0515 mg/0.2ml

Concentration in 1ml = 5 x 0.0515mg/0.2ml

= 1.288mg/ml

Therefore in 100ml = 1.288 x 100

= 129 mg/100ml

Absorbance for sample B = 0.310

Hence concentration in sample B = 0.052mg/0.2ml

Therefore in 100ml = 130mg/100ml

Concentration in sample using formula;

Glucose concentration A = 100 x (absorbance of sample/Absorbance of glucose standard)

= 100 x (0.306/0.166)

= 184.3 mg/100m

Concentration of Sample B = 100 x (0.310/0.166)

= 187mg/100ml
Chemical equations;

Glucose + Cu2+ Cu+ + Gluconic Acid (conditions; OH- and heat)

2Cu+ + MO3+ 2Cu2+ + MO4+

1) Barrette, K., Boitano, S., Barman, S.,Brooks, H. (2012). Ganong’s Review of Medical

Physiology. 24th edition. (McGraw-Hill Education: Singapore) pg 449

2) Devlin, T. (2010) Textbook of Biochemistry With Clinical Correlations. 7th edition.

(John Wiley and Sons, Inc: New York, USA) pgs 849-855

3) Harvey, R., Champe, P and Ferner, D. (2006) Lippincott’s Illustrated Review

Biochemistry. 3rd edition. (Lippincott Williams an Wilkins Company: New York,

USA) pg 95

4) Murray, R., Granner, D., Mayes, P. and Rodwell, V. (2003) Harpers’s Illustrated

Biochemistry. 26th edition. (The McGraw-Hill Companies, Inc: New York, USA) pg

112

5) Nelson, D. and Cox, M. (2004) Lehninger Principles of Biochemistry. 4th edition. (W.

H. Freeman: New York, USA) pgs 238